Anti-VEGF antibody compositions and methods

ABSTRACT

Disclosed are human antibodies that specifically inhibit VEGF binding to only one (VEGFR2) of the two primary VEGF receptors. The antibodies effectively inhibit angiogenesis and induce tumor regression, and yet have improved safety due to their specificity. The present invention thus provides new human antibody-based compositions, methods and combined protocols for treating cancer and other angiogenic diseases. Advantageous immunoconjugate compositions and methods using the new VEGF-specific human antibodies are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. Ser. No.13/225,792, filed Sep. 6, 2011, now U.S. Pat. No. 8,394,943, issued Mar.12, 2013, which is a divisional of U.S. Ser. No. 12/267,515, filed Nov.7, 2008, now U.S. Pat. No. 8,034,905, issued Oct. 11, 2011, which claimspriority to first U.S. provisional application Ser. No. 60/987,015,filed Nov. 9, 2007, second U.S. provisional application Ser. No.61/106,047, filed Oct. 16, 2008, and third U.S. provisional applicationSer. No. 61/108,023, filed Oct. 24, 2008, the entire specification,claims, sequences and drawings of which are incorporated herein byreference without disclaimer.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled1181312SequenceListing.txt, was created on Feb. 14, 2013 and is 32 kb insize. The information in the electronic format of the Sequence Listingis part of the present application and is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of antibodies,angiogenesis and tumor treatment. More particularly, it provides humananti-VEGF antibodies that specifically inhibit VEGF binding to only one(VEGFR2) of the two VEGF receptors. Such antibodies are designed toinhibit angiogenesis and induce tumor regression, and yet have improvedsafety due to their specific blocking properties. The antibody-basedcompositions and methods of the invention also extend to the use ofimmunoconjugates and other therapeutic combinations, kits and methods.

2. Description of the Related Art

Tumor cell resistance to chemotherapeutic agents represents asignificant problem in clinical oncology. In fact, this is one of themain reasons why many of the most prevalent forms of human cancer stillresist effective chemotherapeutic intervention, despite certain advancesin this field.

Another tumor treatment strategy is the use of an “immunotoxin”, inwhich an anti-tumor cell antibody is used to deliver a toxin to thetumor cells. However, in common with chemotherapeutic approaches,immunotoxin therapy also suffers from significant drawbacks when appliedto solid tumors. For example, antigen-negative or antigen-deficientcells can survive and repopulate the tumor or lead to furthermetastases.

A further reason for solid tumor resistance to antibody-based therapiesis that the tumor mass is generally impermeable to macromolecular agentssuch as antibodies and immunotoxins (Burrows et al., 1992; Dvorak etal., 1991a; Baxter and Jain, 1991). Both the physical diffusiondistances and the interstitial pressure within the tumor are significantlimitations to this type of therapy. Therefore, solid tumors, which makeup over 90% of all human cancers, have thus far proven resistant toantibody and immunotoxin treatment.

A more recent strategy has been to target the vasculature of solidtumors. Targeting the blood vessels of the tumors, rather than the tumorcells themselves, has certain advantages in that it is not likely tolead to the development of resistant tumor cells, and that the targetedcells are readily accessible. Moreover, destruction of the blood vesselsleads to an amplification of the anti-tumor effect, as many tumor cellsrely on a single vessel for their oxygen and nutrients (Burrows andThorpe, 1993; 1994). Exemplary vascular targeting strategies aredescribed in U.S. Pat. Nos. 5,855,866 and 5,965,132, which particularlydescribe the targeted delivery of anti-cellular agents and toxins tomarkers of tumor vasculature.

Another effective version of the vascular targeting approach is totarget a coagulation factor to a marker expressed or adsorbed within thetumor vasculature (Huang et al., 1997; U.S. Pat. Nos. 5,877,289,6,004,555, and 6,093,399). The delivery of coagulants, rather thantoxins, to tumor vasculature has the further advantages of reducedimmunogenicity and even lower risk of toxic side effects. As disclosedin U.S. Pat. No. 5,877,289, a preferred coagulation factor for use insuch tumor-specific “coaguligands” is a truncated version of the humancoagulation-inducing protein, Tissue Factor (TF), the major initiator ofblood coagulation.

Although the specific delivery of toxins and coagulation factors totumor blood vessels represents a significant advance in tumor treatment,certain peripheral tumor cells can survive the intratumoral destructioncaused by such therapies. Anti-angiogenic strategies would therefore beof use in combination with the tumor destruction methods of U.S. Pat.Nos. 5,855,866 and 6,004,555.

Anti-angiogenic tumor treatment strategies are based upon inhibiting theproliferation of budding vessels, generally at the periphery of a solidtumor. These therapies are particularly effective in reducing the riskof micrometastasis and inhibiting growth of a solid tumor after, or inconjunction with, more conventional intervention (such as surgery orchemotherapy).

Angiogenesis is the development of new vasculature from preexistingblood vessels and/or circulating endothelial stem cells (Asahara et al.,1997; Springer et al., 1998; Folkman and Shing, 1992). Angiogenesisplays a vital role in many physiological processes, such asembryogenesis, wound healing and menstruation. Angiogenesis is alsoimportant in certain pathological events. In addition to a role in solidtumor growth and metastasis, other notable conditions with an angiogeniccomponent are arthritis, psoriasis and diabetic retinopathy (Hanahan andFolkman, 1996; Fidler and Ellis, 1994).

Angiogenesis is regulated in normal and malignant tissues by the balanceof angiogenic stimuli and angiogenic inhibitors that are produced in thetarget tissue and at distant sites (Fidler et al., 1998; McNamara etal., 1998). Vascular endothelial growth factor-A (VEGF, also known asvascular permeability factor, VPF) is a primary stimulant ofangiogenesis. VEGF is a multifunctional cytokine that is induced byhypoxia and oncogenic mutations and can be produced by a wide variety oftissues (Kerbel et al., 1998; Mazure et al., 1996).

The recognition of VEGF as a primary stimulus of angiogenesis inpathological conditions has led to various attempts to block VEGFactivity Inhibitory anti-VEGF receptor antibodies, soluble receptorconstructs, antisense strategies, RNA aptamers against VEGF and lowmolecular weight VEGF receptor tyrosine kinase (RTK) inhibitors have allbeen proposed for use in interfering with VEGF signaling (Siemeister etal., 1998). Following the inhibition of tumor growth in mice using amurine antibody, (Kim et al., 1993; Asano et al., 1998; Mesiano et al.,1998; Luo et al., 1998a; 1998b; Borgstrom et al., 1996; 1998), ahumanized anti-VEGF antibody termed Avastin (bevacizumab) (Presta etal., 1997) has been approved for clinical use (Hurwitz et al., 2004).

Other murine antibodies that recognize VEGF and inhibit VEGF-inducedfunctions have been reported. These include the murine antibody termed2C3, which has the advantage of inhibiting VEGF binding to only one ofthe two primary VEGF receptors (Brekken et al., 2000). By blocking VEGFbinding to VEGFR2, but not VEGFR1, the murine 2C3 antibody has animproved safety profile, maintaining beneficial effects mediated viaVEGFR1 (Brekken et al., 2000; U.S. Pat. Nos. 6,342,219, 6,524,583,6,342,221).

The inventors have recognized, however, that the identification ofadditional agents that recognize VEGF and inhibit VEGF-inducedangiogenesis would be of benefit in expanding the number of therapeuticoptions. For example, the murine 2C3 antibody, although promising, hascertain limitations. In particular, the 2C3 antibody does not bind tomouse VEGF, meaning that it cannot be used in preclinical studies usingmouse syngeneic tumors. The most effective translation from preclinicalstudies to clinical use would thus benefit from the development of a newantibody that binds to both mouse and human VEGF.

In addition, the inventors have recognized that the development oftherapeutic agents for the treatment of humans that are better toleratedfrom an immunological perspective would be advantageous. In this regard,human antibodies generally have at least three potential advantages foruse in human therapy. First, the human immune system should notrecognize the antibody as foreign. Second, the half-life in the humancirculation will be similar to naturally occurring human antibodies,allowing smaller and less frequent doses to be given. Third, because theeffector portion is human, it will interact better with the other partsof the human immune system, e.g., to destroy target cells moreefficiently by complement-dependent cytotoxicity (CDC) orantibody-dependent cellular cytotoxicity (ADCC).

However, although human antibodies are generally recognized to displaythese advantages, it is known that the development of human antibodiesthat have high enough affinities and appropriate functional propertiesto make them candidates for successful human therapy is by no meansstraightforward. The art therefore still lacks agents that inhibitVEGF-induced angiogenesis for the safe and effective treatment ofhumans, and poses challenges to the development of such agents.

SUMMARY OF THE INVENTION

The present invention overcomes certain limitations in the prior art byproviding new therapeutic compositions and methods for use inanti-angiogenic and anti-tumor treatment. The invention is based onhuman antibodies that have the functional property of specificallyinhibiting VEGF binding to only one (VEGFR2) of the two primary VEGFreceptors, and have an affinity for VEGF high enough for effectivetreatment regimens. Such antibodies inhibit angiogenesis and treattumors as effectively as other anti-VEGF antibodies, including thosealready approved for clinical use, and yet have improved safety due totheir specific blocking properties. The compositions and methods of theinvention also extend to the use of immunoconjugates and combinations,including prodrugs, using the specific category of antibodies provided.

A particular advantage of the present invention is that the humanantibodies provided inhibit VEGF binding only to VEGFR2, and not VEGFR1.This contrasts with the leading antibodies in the prior art, includingA4.6.1 and the humanized version, Avastin, which inhibit VEGF binding toboth VEGFR2 and VEGFR1. As VEGFR1 has important biological rolesunconnected to angiogenesis, e.g., in osteoclast and chondroclastfunction, the present ability to inhibit only VEGFR2-mediatedangiogenesis is a distinct advantage. This translates into notableclinical benefits in that bone metabolism, e.g., in the treatment ofpediatric cancers, is not adversely affected. The harmful effects ofmacrophages on tumor progression and metastasis are also inhibited, asthis population of macrophages expresses VEGFR2, which is inhibited bythe antibodies of the invention.

A further advantage is that, as binding of VEGF to VEGFR1 is maintainedin the presence of the antibodies of the invention, they can be used tospecifically deliver attached therapeutic agents to tumor vasculature byvirtue of binding to VEGF that is bound to VEGFR1, which is upregulatedon tumor endothelium. In the context of immunoconjugates, therefore, thepresent invention provides agents that have both anti-angiogenic andtumor destructive properties within the same molecule.

Yet a further advantage exists in the ability of the compositionsprovided to neutralize the survival signal of VEGF, which is mediatedthrough VEGFR2. The naked and conjugated antibodies of the inventionthus form synergistic combinations with other therapies and/or attachedagents, particularly those methods and agents that fail to achievemaximal effectiveness in vivo due to the ability of VEGF to counteracttheir destructive properties.

Amino acid and/or DNA sequences of antibody molecules of the inventionthat bind to VEGF, their V_(H) and V_(L) domains includingcomplementarity determining regions (CDRs), are set forth in the variousSEQ ID NOs. listed herein.

In one embodiment, the present invention provides an antibody that bindsto VEGF comprising a heavy chain CDR1 domain comprising the amino acidsequence of SEQ ID NO:5 or a sequence substantially homologous thereto.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to VEGF comprises a heavy chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO:6 or a sequencesubstantially homologous thereto.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to VEGF comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO:7 or a sequencesubstantially homologous thereto.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to VEGF comprises a light chain CDR1 domaincomprising the amino acid sequence of SEQ ID NO:8 or a sequencesubstantially homologous thereto.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to VEGF comprises a light chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO:9 or a sequencesubstantially homologous thereto.

Alternatively or in addition, in an embodiment of the invention, theantibody that binds to VEGF comprises a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO:10 or a sequencesubstantially homologous thereto.

Thus, in certain embodiments, the invention provides an antibody thatbinds to VEGF comprising one or more heavy chain CDR domains, whereinthe heavy chain CDR domain is selected from the group consisting of:

(a) a heavy chain CDR1 domain comprising the amino acid sequence of SEQID NO:5 or a sequence substantially homologous thereto;

(b) a heavy chain CDR2 domain comprising the amino acid sequence of SEQID NO:6 or a sequence substantially homologous thereto; and

(c) a heavy chain CDR3 domain comprising the amino acid sequence of SEQID NO:7 or a sequence substantially homologous thereto.

The invention also provides, in certain embodiments an antibody thatbinds to VEGF comprising one or more light chain CDR domains, whereinthe light chain CDR domain is selected from the group consisting of:

(a) a light chain CDR1 domain comprising the amino acid sequence of SEQID NO:8 or a sequence substantially homologous thereto;

(b) a light chain CDR2 domain comprising the amino acid sequence of SEQID NO:9 or a sequence substantially homologous thereto; and

(c) a light chain CDR3 domain comprising the amino acid sequence of SEQID NO:10 or a sequence substantially homologous thereto.

In certain preferred embodiments, the antibody that binds to VEGFcomprises both

(a) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:7or a sequence substantially homologous thereto and

(b) a light chain CDR3 comprising the amino acid sequence of SEQ IDNO:10 or a sequence substantially homologous thereto.

More preferably, a heavy chain CDR1 domain comprising the amino acidsequence of SEQ ID NO:5 or a sequence substantially homologous theretoand/or a light chain CDR1 domain comprising the amino acid sequence ofSEQ ID NO:8 or a sequence substantially homologous thereto, and/or aheavy chain CDR2 domain comprising the amino acid sequence of SEQ IDNO:6 or a sequence substantially homologous thereto and/or a light chainCDR2 domain comprising the amino acid sequence of SEQ ID NO:9 or asequence substantially homologous thereto, are also present.

In one preferred embodiment, the heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO:5 or a sequence substantially homologousthereto, CDR2 comprising the amino acid sequence of SEQ ID NO:6 or asequence substantially homologous thereto, and CDR3 comprising the aminoacid sequence of SEQ ID NO:7, or a sequence substantially homologousthereto, are present individually or in combination.

In yet another preferred embodiment, the light chain CDR1 comprising theamino acid sequence of SEQ ID NO:8 or a sequence substantiallyhomologous thereto, CDR2 comprising the amino acid sequence of SEQ IDNO:9 or a sequence substantially homologous thereto, and CDR3 comprisingthe amino acid sequence of SEQ ID NO:10 or a sequence substantiallyhomologous thereto, are present individually or in combination.

Viewed alternatively, in certain embodiments, the present inventionprovides an antibody that binds to VEGF comprising

a heavy chain CDR3 domain comprising the amino acid sequence of SEQ IDNO:7 or a sequence substantially homologous thereto and/or a light chainCDR3 domain comprising the amino acid sequence of SEQ ID NO:10 or asequence substantially homologous thereto. Said antibody optionallyfurther comprisesa heavy chain CDR2 domain comprising the amino acid sequence of SEQ IDNO:6 or a sequence substantially homologous thereto and/or a light chainCDR2 domain comprising the amino acid sequence of SEQ ID NO:9 or asequence substantially homologous thereto and/or further comprises aheavy chain CDR1 domain comprising the amino acid sequence of SEQ IDNO:5 or a sequence substantially homologous thereto and/ora light chain CDR1 domain comprising the amino acid sequence of SEQ IDNO:8 or a sequence substantially homologous thereto.

Viewed alternatively, in certain embodiments, the present inventionprovides an antibody that binds to VEGF comprising

a heavy chain CDR2 domain comprising the amino acid sequence of SEQ IDNO:6 or a sequence substantially homologous thereto and/or a light chainCDR2 domain comprising the amino acid sequence of SEQ ID NO:9 or asequence substantially homologous thereto.

Said antibody optionally further comprises

a heavy chain CDR3 domain comprising the amino acid sequence of SEQ IDNO:7 or a sequence substantially homologous thereto and/or a light chainCDR3 domain comprising the amino acid sequence of SEQ ID NO:10 or asequence substantially homologous thereto and/or further comprises aheavy chain CDR1 domain comprising the amino acid sequence of SEQ IDNO:5 or a sequence substantially homologous thereto and/or a light chainCDR1 domain comprising the amino acid sequence of SEQ ID NO:8 or asequence substantially homologous thereto.

Viewed alternatively, in certain embodiments, the present inventionprovides an antibody that binds to VEGF comprising a heavy chain CDR1domain comprising the amino acid sequence of SEQ ID NO:5 or a sequencesubstantially homologous thereto and/or a light chain CDR1 domaincomprising the amino acid sequence of SEQ ID NO:8 or a sequencesubstantially homologous thereto. Said antibody optionally furthercomprises a heavy chain CDR3 domain comprising the amino acid sequenceof SEQ ID NO:7 or a sequence substantially homologous thereto and/or alight chain CDR3 domain comprising the amino acid sequence of SEQ IDNO:10 or a sequence substantially homologous thereto and/or furthercomprises a heavy chain CDR2 domain comprising the amino acid sequenceof SEQ ID NO:6 or a sequence substantially homologous thereto and/or alight chain CDR2 domain comprising the amino acid sequence of SEQ IDNO:9 or a sequence substantially homologous thereto.

Certain preferred antibodies of the invention comprise one or more ofthe CDRs selected from the group consisting of SEQ ID NOs:5, 6, 7, 8, 9and 10 or a sequence substantially homologous to any one of theforegoing SEQ ID NOs.

Certain preferred antibodies comprise two or more of the light chainCDRs of SEQ ID NOs:8, 9 or 10, or sequences substantially homologous toany one of the foregoing SEQ ID NOs. Especially preferred bindingmolecules comprise 3 of the light chain CDRs of SEQ ID NOs:8, 9 or 10,or sequences substantially homologous to any one of the foregoing SEQ IDNOs (i.e. one of each of the aforementioned light chain CDR1 and CDR2and CDR3 or sequences substantially homologous thereto).

Other certain preferred antibodies comprise two or more of the heavychain CDRs of SEQ ID NOs:5, 6 or 7, or sequences substantiallyhomologous to any one of the foregoing SEQ ID NOs. Especially preferredbinding molecules comprise 3 of the heavy chain CDRs of SEQ ID NOs:5, 6and 7, or sequences substantially homologous to any one of the foregoingSEQ ID NOs (i.e., one of each of the aforementioned heavy chain CDR1 andCDR2 and CDR3 or sequences substantially homologous thereto).

Certain more especially preferred antibodies comprise 3 of the lightchain CDRs of SEQ ID NOs:8, 9 or 10 or sequences substantiallyhomologous to any one of these sequences (i.e., one of each of theaforementioned light chain CDR1 and CDR2 and CDR3 or sequencessubstantially homologous thereto), and 3 of the heavy chain CDRs of SEQID NOs:5, 6 or 7, or sequences substantially homologous any one of thesesequences (i.e., one of each of the aforementioned heavy chain CDR1 andCDR2 and CDR3 or sequences substantially homologous thereto).

Certain especially preferred antibodies comprise a heavy chain CDR1domain of SEQ ID NO:5, a heavy chain CDR2 domain of SEQ ID NO:6, and aheavy chain CDR3 domain of SEQ ID NO:7, or sequences substantiallyhomologous to any one of the aforementioned sequences; and/or comprise alight chain CDR1 domain of SEQ ID NO:8, a light chain CDR2 domain of SEQID NO:9, and a light chain CDR 3 domain of SEQ ID NO:10, or sequencessubstantially homologous to any one of the aforementioned sequences.

In a further embodiment, the invention provides an antibody that bindsto VEGF and that comprises at least one heavy chain variable region thatcomprises three CDRs and at least one light chain variable region thatcomprises three CDRs, wherein said light chain variable regioncomprises:

(i) a variable light (VL) CDR1 that has the amino acid sequence of SEQID NO:8,

(ii) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9, and

(iii) a VL CDR3 that has the amino acid sequence of SEQ ID NO:10.

In a preferred aspect of this embodiment, one or more of said heavychain variable region CDRs are selected from the group consisting of:

(i) a VH CDR1 that has the amino acid sequence of SEQ ID NO:5,

(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO:6, and

(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO:7.

In a further preferred aspect of this embodiment, two of said heavychain variable region CDRs are selected from the group consisting of:

(i) a VH CDR1 that has the amino acid sequence of SEQ ID NO:5,

(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO:6, and

(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO:7.

In a yet further preferred aspect of this embodiment, three of saidheavy chain variable region CDRs are selected from the group consistingof:

(i) a VH CDR1 that has the amino acid sequence of SEQ ID NO:5,

(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO:6, and

(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO:7.

Certain further preferred embodiments of the invention provide anantibody that binds to VEGF and that comprises: a VH domain thatcomprises one, two or three of the heavy chain CDRs of SEQ ID NOs:5, 6,or 7, or sequences substantially homologous to one or more of SEQ IDNOs:5, 6, or 7, and/or a VL domain that comprises one, two or three ofthe light chain CDRs of SEQ ID NOs:8, 9 or 10, or sequencessubstantially homologous to one or more of SEQ ID NOs:8, 9 or 10.

Especially preferred VL domains comprise 3 of the light chain CDRs ofSEQ ID NOs:8, 9 and 10, or sequences substantially homologous to one ormore of SEQ ID NOs:8, 9 or 10, (i.e., one of each of CDR1, CDR2 and CDR3or sequences substantially homologous thereto).

Especially preferred VH domains comprise 3 of the heavy chain CDRs ofSEQ ID NOs:5, 6, and 7, or sequences substantially homologous to one ormore of SEQ ID NOs:5, 6, or 7 (i.e., one of each of CDR1, CDR2 and CDR3or sequences substantially homologous thereto).

More especially preferred embodiments of the invention provide anantibody that binds to VEGF and that comprises: a VL domain thatcomprises 3 light chain CDRs of SEQ ID NOs:8, 9 and 10, and a VH domainthat comprises 3 heavy chain CDRs. In preferred embodiments one, two orthree of the heavy chain CDRs are as defined in SEQ ID NOs:5, 6, and 7.

Certain preferred embodiments of the invention provide an antibody thatbinds VEGF comprising a VH domain that has the amino acid sequence ofSEQ ID NO:3 or a sequence substantially homologous thereto and/or a VLdomain that has the amino acid sequence of SEQ ID NO:4 or a sequencesubstantially homologous thereto.

Further preferred embodiments provide an antibody that binds VEGFcomprising a VL domain that has the amino acid sequence of SEQ ID NO:4and a VH domain that comprises 3 heavy chain CDRs. Preferably said VHdomain has the amino acid sequence of SEQ ID NO:3.

In a yet further embodiment, the present invention provides an antibodythat binds VEGF comprising the amino acid sequence of SEQ ID NO:21 (saidantibody also being referred to herein as r84 or PGN311 orEJ173/112-C11), or comprising a fragment thereof that binds VEGF, or asequence substantially homologous thereto.

In a further embodiment, the present invention provides an antibody thatbinds VEGF comprising the amino acid sequence of SEQ ID NO:21 (saidantibody also being referred to herein as r84 or PGN311 orEJ173/112-C11), or comprising a fragment thereof that binds VEGF.

The invention is exemplified by monoclonal antibody r84 (also referredto herein as PGN311 and EJ-173-112-C11), a single chain form of which isshown in FIG. 1 (SEQ ID NO:21 and SEQ ID NO:20) and a full length IgGform of which is shown in Example 6. The CDR domains, VH and VL domainsof the r84 antibody are shown in Table 1 and FIG. 1. Antibodiescomprising these CDR domains or VH and VL domains (or sequencessubstantially homologous thereto) are preferred aspects of theinvention.

A preferred embodiment of the invention is a scFv form of the r84antibody shown in SEQ ID NO:21 (amino acid), which is preferably encodedby SEQ ID NO:20 (nucleic acid).

Another preferred embodiment of the invention is a full length IgG formof the r84 antibody, the heavy chain of which is shown in SEQ ID NO:24(amino acid), which is preferably encoded by SEQ ID NO:22 (nucleicacid); and the light chain of which is shown in SEQ ID NO:25 (aminoacid), which is preferably encoded by SEQ ID NO:23 (nucleic acid).

Certain examples of substantially homologous sequences are sequencesthat have at least 70% identity to the amino acid sequences disclosed.

In certain embodiments, the antibodies of the invention that bind toVEGF comprise at least one light chain variable region that includes anamino acid sequence region of at least about 75%, more preferably atleast about 80%, more preferably at least about 85%, more preferably atleast about 90% or 95% and most preferably at least about 97%, 98% or99% amino acid sequence identity to the amino acid sequence of SEQ IDNO:4; and/or at least one heavy chain variable region that includes anamino acid sequence region of at least about 75%, more preferably atleast about 80%, more preferably at least about 85%, more preferably atleast about 90% or 95% and most preferably at least about 97%, 98% or99% amino acid sequence identity to the amino acid sequence of SEQ IDNO:3.

Other preferred examples of substantially homologous sequences aresequences containing conservative amino acid substitutions of the aminoacid sequences disclosed.

Other preferred examples of substantially homologous sequences aresequences containing 1, 2 or 3, preferably 1 or 2, altered amino acidsin one or more of the CDR regions disclosed. Such alterations might beconserved or non-conserved amino acid substitutions, or a mixturethereof.

In all such embodiments, preferred alterations are conservative aminoacid substitutions.

In all embodiments, the antibodies containing substantially homologoussequences retain the ability to bind VEGF.

In embodiments of the invention where alterations in the light chainCDR3 domain are contemplated, it is preferred that the L residue atposition 8 in said CDR is retained without variation.

Other embodiments of the present invention provide binding proteins thatbind to VEGF and that comprise an antibody of the invention, a VH or VLdomain of the invention, or one or more of the CDRs of the invention. Ina preferred embodiment, such binding proteins are antibodies.

Preferred antibodies of the invention comprise at least one heavy chainvariable region that comprises three CDRs and at least one light chainvariable region that comprises three CDRs. Exemplary and preferredsequences for these CDRs are described herein.

As used herein, the succinct term “VEGF”, unless otherwise specificallystated or made clear from the scientific terminology, means VascularEndothelial Growth Factor-A (VEGF-A), also known as vascularpermeability factor, VPF.

“VEGF” also means any form of VEGF, particularly as VEGF is conservedacross mammalian species. The antibodies or antibody fragments of theinvention may thus bind to human, monkey, cow (bovine), mouse, rat,hamster, ferret, guinea pig and/or rabbit VEGF, for example. Preferably,the antibodies or antibody fragments of the invention will bind at leastto human VEGF. In certain preferred embodiments, the antibodies orantibody fragments of the invention will bind at least to human andmouse VEGF.

As used herein, the term “that binds VEGF” in the context of antibodiesor antibody fragments of the present invention, means human antibodiesor antibody fragments that are capable of one or more of the following;preferably, of more than one of the following; and most preferably, ofall of the following:

-   -   (a) bind to a non-conformationally dependent VEGF epitope, as        assessed by binding to VEGF in a Western blot;    -   (b) bind to free VEGF or to VEGF on a solid support;    -   (c) bind at least to human VEGF and mouse VEGF;    -   (d) significantly inhibit or significantly reduce VEGF binding        to the VEGF receptor VEGFR2 (KDR/Flk-1);    -   (e) do not significantly inhibit VEGF or reduce binding to the        VEGF receptor VEGFR1 (Flt-1);    -   (f) inhibit, and preferably, significantly inhibit, VEGF-induced        phosphorylation of VEGFR2;    -   (g) inhibit, and preferably, significantly inhibit, VEGF-induced        vascular permeability;    -   (h) inhibit, and preferably, significantly inhibit,        VEGF-mediated endothelial cell proliferation;    -   (i) inhibit, and preferably, significantly inhibit,        angiogenesis;    -   (j) inhibit, and preferably, significantly inhibit,        lymphangiogenesis;    -   (k) do not significantly inhibit VEGFR1-mediated stimulation or        activation of cells, such as VEGFR1-expressing osteoclasts or        chondroclasts; and/or    -   (l) localize to tumor vasculature and/or tumor stroma upon        administration to an animal with a vascularized tumor.

Most preferably, the human antibody or antibody fragment of theinvention is one that significantly inhibits VEGF binding to the VEGFreceptor VEGFR2 (KDR/Flk-1) without significantly inhibiting VEGFbinding to the VEGF receptor VEGFR1 (Flt-1).

The present invention therefore provides human antibodies thatspecifically block VEGF binding to the VEGFR2 receptor, or that blockVEGF binding to essentially only the VEGFR2 receptor. Such humanantibodies significantly inhibit VEGF binding to the VEGFR2 receptor(KDR/Flk-1) without significantly inhibiting VEGF binding to the VEGFR1receptor (Flt-1). Such human antibodies thus inhibit VEGF binding to theVEGFR2 receptor (KDR/Flk-1), do not substantially inhibit VEGF bindingto the VEGFR1 receptor (Flt-1), exhibit anti-angiogenic and anti-tumoreffects in vivo and do not significantly inhibit VEGFR1-mediated events,such as osteoclast or chondroclast functions.

The human antibodies of the invention are thus succinctly termed“VEGFR2-blocking, non-VEGFR1-blocking, human anti-VEGF antibodies”. Evenmore succinctly, they are termed “VEGFR2-blocking, human anti-VEGFantibodies”, which is used for simplicity in reference to allcompositions, uses and methods of the invention. A “VEGFR2-blocking,human anti-VEGF antibody” is a human antibody against VEGF that blocksVEGF binding to the VEGFR2 receptor. It will be clear that suchantibodies are not antibodies against the VEGFR2 receptor itself.

In light of this invention, therefore, a range of VEGFR2-blocking, humananti-VEGF antibodies can be made and used in a variety of embodiments,including in the inhibition of angiogenesis and the treatment ofangiogenic diseases and tumors without inhibiting VEGF signaling via theVEGFR1 receptor and without the notable drawbacks and side effectsassociated therewith.

In certain embodiments, the present application further describesmethodology for generating candidate VEGFR2-blocking, human anti-VEGFantibodies and the routine technical aspects of the assays required toidentify actual VEGFR2-blocking specific antibodies from the pool ofcandidates.

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,except in instances wherein an upper limit is thereafter specificallystated. Therefore, an “antibody”, as used herein, means “at least afirst antibody”. The operable limits and parameters of combinations, aswith the amounts of any single agent, will be known to those of ordinaryskill in the art in light of the present disclosure.

Human antibodies of the invention that “specifically inhibit VEGFbinding to the VEGF receptor VEGFR2 (KDR/Flk-1)” can be identified bycompetition and/or functional assays. The preferred assays, forsimplicity, are competition assays based upon an ELISA. In competitionassays, one pre-mixes or admixes VEGF with varying amounts of the testantibodies (e.g., 100-fold to 1000-fold molar excess, e.g., 500-fold or750-fold molar excess) and determines the ability of the test antibodiesto reduce VEGF binding to VEGFR2. VEGF can be pre-labeled and detecteddirectly, or can be detected using a (secondary) anti-VEGF antibody or asecondary and tertiary antibody detection system. An ELISA format ofsuch a competition assay is a preferred format, but any type ofimmunocompetition assay may be conducted.

VEGF binding to VEGFR2 in the presence of a completely irrelevantantibody (including non-blocking anti-VEGF monoclonal antibodies) is thecontrol high value (100%) in such a competition assay. In a test assay,a significant reduction in VEGF binding to VEGFR2 in the presence of atest antibody is indicative of a test antibody that significantlyinhibits VEGF binding to the VEGF receptor VEGFR2 (KDR/Flk-1).

A significant reduction is a “reproducible”, i.e., consistentlyobserved, reduction in binding. A “significant reduction” in terms ofthe present application is defined as a reproducible reduction (in VEGFbinding to VEGFR2) of at least about 50%, about 55%, about 60% or about65% at any amount between about 100 fold and about 1000 fold (e.g.,about 500 fold or about 750 fold) molar excess of antibody over VEGF.Viewed alternatively a signal of less than 50% (when compared to acontrol value of 100%) is considered significant inhibition of binding.

A preferred feature of the invention is that the human antibodiesprovided do not substantially or significantly inhibit, reduce or blockVEGF binding to VEGFR1. Human antibodies that exhibit a moderatelysignificant reduction of VEGF binding to VEGFR2 will still be useful, solong as they do not substantially inhibit VEGF binding to VEGFR1.Nonetheless, more preferred antibodies will be those that have a moresignificant ability to inhibit VEGF binding to VEGFR2. These antibodiesare those that exhibit a reproducible ability to reduce VEGF binding toVEGFR2 by at least about 70%, about 75% or about 80% at any amountbetween about 100 fold and about 1000 fold (e.g., about 500 fold orabout 750 fold) molar excess of antibody over VEGF. Although notrequired to practice the invention, antibodies that reduce VEGF bindingto VEGFR2 by at least about 85%, about 90%, about 95% or even higher areby no means excluded.

Human anti-VEGF antibodies, or antigen-binding fragments thereof, thatinhibit VEGF binding to the VEGF receptor VEGFR2 (KDR/Flk-1) withoutsignificantly inhibiting VEGF binding to the VEGF receptor VEGFR1(Flt-1) are readily confirmed by simple competition assays such as thosedescribed above, but using VEGFR1.

Absence of a significant inhibition or reduction is a “reproducible”,i.e., consistently observed, “substantial maintenance of binding”. A“substantial maintenance of binding” in terms of the present applicationis defined as a reproducible maintenance (in VEGF binding to VEGFR1) ofat least about 60%, about 75%, about 80% or about 85% at any amountbetween about 100 fold and about 1000 fold molar excess of antibody overVEGF.

The intention of using human antibodies that do not substantiallyinhibit VEGF binding to VEGFR1 is to maintain the biological functionsmediated by VEGFR1. Therefore, an antibody need only maintain sufficientVEGF binding to VEGFR1 so that a biological response is induced by VEGF.Nonetheless, more preferred antibodies will be those that have a moresignificant ability to maintain VEGF binding to VEGFR1. These antibodiesare those that exhibit a reproducible ability to maintain VEGF bindingto VEGFR1 at levels of at least about 88%, about 90%, about 92%, about95% or of about 98-99% at any amount between about 100 fold and about1000 fold molar excess of antibody over VEGF.

It will be understood that human antibodies that more substantiallyinhibit VEGF binding to VEGFR2 can likely tolerate more reduction inbinding VEGFR1. Equally, where an antibody has a moderate reduction inVEGF binding to VEGFR2, the maintenance of binding to VEGFR1 should bemore stringently pursued.

Another preferred binding assay to identify and/or confirm that anantibody inhibits VEGF binding to the VEGF receptor VEGFR2 (KDR/Flk-1)is a co-precipitation assay. A co-precipitation assay tests the abilityof an antibody to block the binding of VEGF to one or more receptors insolution. In such an assay, VEGF or detectably-labeled VEGF is incubatedwith a suitable form of the receptor.

There are many formats for conducting immunoprecipitation orco-precipitation assays. In the present case, a “suitable form of thereceptor” may be the VEGFR2 receptor at issue or the extracellulardomain of the receptor Immunoprecipitation will then require, as well asthe standard reagents, the presence of an antibody against the VEGFR2receptor or an epitope on the extracellular domain of the receptordistinct from the site to which VEGF binds. The present inventionprovides other “suitable” forms of the VEGF receptors, namely theextracellular domains of the receptors linked to an Fc antibody portion.Such receptor/Fc constructs can be precipitated by incubation with aneffective immunoprecipitating composition, such as a Protein A-basedcomposition.

Irrespective of the suitable receptor, the immunoprecipitation orco-precipitation assays are preferably conducted with controls. Theability of VEGF alone to bind to the chosen receptor should be confirmedby precipitation in the absence of an anti-VEGF antibody. Preferably,parallel incubations are conducted in the presence or absence of anantibody with known binding properties to act as a control. Mostpreferably, assays using both a blocking control and non-blockingcontrol antibody are run in parallel.

Any bound immunological species are then immunoprecipitated, e.g., byincubation with an effective immunoprecipitating composition, such as aProtein A composition or Protein A sepharose beads. The precipitate isthen tested for the presence of VEGF. Where the VEGF in the initialincubation was detectably-labeled VEGF, such as radio-labeled VEGF, anyVEGF in the immunoprecipitates can be detected directly. Any non-labeledVEGF in the immunoprecipitates may be detected by other suitable means,e.g., by gel separation and immunodetection with an anti-VEGF antibody.

The ability of a human antibody to block VEGF binding to a VEGFreceptor, such as VEGFR2, in such a co-precipitation assay can bereadily quantitated, although qualitative results are also valuable.Quantification can be achieved by direct measurement of labeled VEGF ore.g., by densitometric analyses of immunodetected VEGF. Antibodies thatexhibit a reproducible, i.e., consistently observed, ability to inhibitVEGF binding to VEGFR2 can thus be detected, and useful antibodies canbe chosen according to the quantitative criteria outlined above.

Human anti-VEGF antibodies that do not significantly inhibit VEGFbinding to the VEGF receptor VEGFR1 (Flt-1) can also be readilyidentified by conducting co-precipitation assays as described above, butusing VEGFR1 rather than VEGFR2. Therefore, anti-VEGF antibodies thatinhibit VEGF binding to the VEGF receptor VEGFR2 (KDR/Flk-1) withoutsignificantly inhibiting VEGF binding to the VEGF receptor VEGFR1(Flt-1) can also be readily identified using such methods.

The present application also provides various functional assays toidentify and/or confirm that a human antibody significantly inhibitsVEGF binding to the VEGF receptor VEGFR2 (KDR/Flk-1). These aregenerally related to the identification of VEGFR2 as the receptorresponsible for certain defined biological responses. Although theforegoing competition-type assays, which are conducted in cell-freesystems, are most reproducible, reliable, labor-saving andcost-effective, the following assays are also useful in the context ofthe present invention.

For example, a VEGFR2-blocking, human anti-VEGF antibody may beidentified by testing for the ability to inhibit VEGF-mediatedendothelial cell growth (inhibiting the mitogenic activity of VEGF). Anysuitable assay may be employed using any of a variety of endothelialcells in the presence of VEGF with or without test antibodies.Preferably, duplicate assays are run in parallel, such as those withoutVEGF and those with control antibodies of defined properties (bothblocking and non-blocking). Endothelial cell growth may be determinedand preferably accurately quantified by any acceptable means ofdetermining cell number, including colorimetric assays.

A human antibody with an ability to inhibit VEGF-mediated endothelialcell growth will generally exhibit a consistently observed inhibition ofVEGF-mediated endothelial cell growth of about 25%, 30%, 35%, 40% 45% or50% or so Inhibition in such ranges will indicate an antibody withproperties sufficient to inhibit angiogenesis in vivo. Antibodies withmore significant inhibitory activity are not excluded from theinvention.

Further functional assays to identify human antibodies in accordancewith the present invention are assays to test blocking of VEGF-inducedphosphorylation. Any suitable assay may be employed using any of avariety of endothelial cells that express any form of native orrecombinant phosphorylatable VEGFR2. Cells are incubated with VEGF inthe presence or absence of the antibody to be tested for a suitable timeperiod. Preferably, duplicate assays are run in parallel, such as thosewithout VEGF and those with control antibodies of defined properties(both blocking and non-blocking).

VEGF-induced phosphorylation of VEGFR2 may be determined and preferablyaccurately quantified by any acceptable means. Generally, VEGFR2 isimmunoprecipitated for further analyses. The degree of phosphorylationof VEGFR2 may be determined directly, for example, the cells may havebeen incubated with ³²P-labelled ATP, allowing direct quantification ofthe ³²P within the immunoprecipitated VEGFR2. Preferably, theimmunoprecipitated VEGFR2 are analyzed by other means, e.g., by gelseparation and immunodetection with an antibody that binds tophosphotyrosine residues. A human antibody with an ability to inhibitVEGF-induced phosphorylation of VEGFR2 will generally exhibit aconsistently observed reduction in the levels of phosphorylated VEGFR2.

Yet further functional assays to identify VEGFR2-blocking, humananti-VEGF antibodies in accordance with the present invention are assaysto test inhibition of VEGF-induced vascular permeability. Although anysuch assay may be used, a particularly suitable assay is the Milespermeability assay, wherein animals such as guinea pigs are injectedwith a dye, such as Evan's blue dye, and the appearance of the dye inthe animal skin is determined after the provision of VEGF in thepresence or absence of test antibodies. Preferably, duplicate studiesare conducted in parallel, such as those without VEGF and those withcontrol antibodies of defined properties (both blocking andnon-blocking). The appearance of dye in the animal skin is typically asspots, such as blue spots, in the back of the animal, which can bephotographed and measured.

VEGFR2-blocking, human anti-VEGF antibodies will inhibitVEGF-induced-vascular permeability as a consistently observed inhibitionat low concentrations, such as when provided at a 100-fold, or 1000-foldmolar excess over VEGF. Antibodies that do not block VEGF binding toVEGFR2 will not show any significant inhibition of VEGF induced-vascularpermeability. Generally, antibodies that block VEGF-induced permeabilityonly at high concentrations, such as at a 10-fold molar excess overVEGF, will not be antibodies with properties in accordance with thepresent invention.

Widely accepted functional assays of angiogenesis and, hence,anti-angiogenic agents are the corneal micropocket assay ofneovascularization and the chick chorio-allantoic membrane assay (CAM)assay. U.S. Pat. No. 5,712,291 is specifically incorporated herein byreference to show that the corneal micropocket and CAM assays aresufficiently predictive to identify agents for use in the treatment ofan extremely wide range of angiogenic diseases.

U.S. Pat. No. 5,001,116 is also specifically incorporated herein byreference for purposes of describing the CAM assay. Essentially,fertilized chick embryos are removed from their shell on day 3 or 4, anda methylcellulose disc containing the test compound is implanted on thechorioallantoic membrane. The embryos are examined approximately 48hours later and, if a clear avascular zone appears around themethylcellulose disc, the diameter of that zone is measured. Asdisclosed in U.S. Pat. No. 5,712,291, specifically incorporated hereinby reference for this purpose, in the context of the present invention,the appearance of any avascular zone is sufficient to evidence ananti-angiogenic antibody. The larger the zone, the more effective theantibody.

The corneal micropocket assay of neovascularization may be practicedusing rat or rabbit corneas. This in vivo model is widely accepted asbeing predictive of clinical usefulness, as evidenced by U.S. Pat. Nos.5,712,291 and 5,871,723, each specifically incorporated herein byreference for evidence purposes. Although not believed to beparticularly relevant the present invention, the corneal assays arepreferable over the CAM assay because they will generally recognizecompounds that are inactive per se but are metabolized to yield activecompounds.

In the present invention, the corneal micropocket assay is used toidentify an anti-angiogenic agent. This is evidenced by a significantreduction in angiogenesis, as represented by a consistently observed andpreferably marked reduction in the number of blood vessels within thecornea. Such responses are preferably defined as those corneas showingonly an occasional sprout and/or hairpin loop that displayed no evidenceof sustained growth when contacted with the test substance.

The invention as claimed is enabled in accordance with the presentspecification and readily available technological references, know-howand starting materials.

Certain preferred embodiments of the invention are thereforecompositions comprising at least a first human anti-VEGF antibody of theinvention, or antigen binding fragment thereof.

Human anti-VEGF antibodies, or antigen-binding fragments thereof, thatspecifically inhibit VEGF binding to the VEGF receptor VEGFR2(KDR/Flk-1); and anti-VEGF antibodies, or antigen-binding fragmentsthereof, that inhibit VEGF binding to the VEGF receptor VEGFR2(KDR/Flk-1) without significantly inhibiting VEGF binding to the VEGFreceptor VEGFR1 (Flt-1) form other aspects of the invention.

Human antibodies with the desired combinations of properties can bereadily identified by one or more or a combination of the receptorcompetition, ELISA, co-precipitation, and/or functional assays describedabove. The guidance concerning the quantitative assessment of antibodiesthat consistently significantly reduce VEGF binding to VEGFR2 and thatconsistently do not significantly inhibit VEGF binding to VEGFR1 is asdescribed above.

r84 is herein shown to reduce the amount of VEGF that bound toVEGFR2-coated ELISA wells to about 11% and 2%, respectively, at 100 foldand 500 fold molar excesses over VEGF. These figures equate toreductions in VEGF binding to VEGFR2 of about 89% and about 98%,respectively. r84 is herein shown to maintain the amount of VEGF thatbound to VEGFR1-coated ELISA wells at about 94% and 84%, respectively,at 100 fold and 500 fold molar excesses over VEGF. Even at 1000 foldmolar excesses over VEGF, r84 still maintains VEGF binding to VEGFR1 atabout 65%. It will again be understood that antibodies that moresubstantially inhibit VEGF binding to VEGFR2 can likely tolerate morereduction in binding VEGFR1. Equally, where an antibody has a moderatereduction in VEGF binding to VEGFR2, the maintenance of binding toVEGFR1 should be more stringently pursued. It will thus be appreciatedthat a “comparative” difference between the two values is important.

Nucleic acid molecules comprising nucleotide sequences that encode thehuman antibodies of the present invention as defined herein or parts orfragments thereof, or nucleic acid molecules substantially homologousthereto, form yet further aspects of the invention. Preferred nucleicacid molecules comprise sequences which encode the amino acid sequenceset out in SEQ ID NO:21 (which is preferably encoded by SEQ ID NO:20).Other preferred nucleic acid molecules comprise sequences which encode aheavy chain that has the amino acid sequence of SEQ ID NO:24 (which ispreferably encoded by SEQ ID NO:22) and encode a light chain which hasthe amino acid sequence of SEQ ID NO:25 (which is preferably encoded bySEQ ID NO:23).

Other preferred nucleic acid molecules comprise sequences that encodeIgG forms of the antibodies of the invention or murine chimeric forms,for example those as described in Example 6.

As indicated above, other nucleic acid molecules encompassed by thepresent invention are those encoding parts or fragments of the humanantibodies of the present invention, e.g., those encoding a heavy chainof an antibody (e.g., those encoding SEQ ID NO:24, such as SEQ ID NO:22)or those encoding a light chain of an antibody (e.g., those encoding SEQID NO:25, such as SEQ ID NO:23). Other preferred nucleic acid moleculesare those encoding a VH region of an antibody of the present invention(e.g., those encoding SEQ ID NO:3, such as SEQ ID NO:1 or SEQ ID NO:26).Other preferred nucleic acid molecules are those encoding a VL region ofan antibody of the present invention (e.g., those encoding SEQ ID NO:4,such as SEQ ID NO:2 or SEQ ID NO:27).

Thus, fragments of the antibodies of the invention as defined herein, orsequences substantially homologous thereto, or nucleic acid moleculescomprising sequences encoding such fragments form a yet further aspectof the invention.

Advantageously, the antibodies of the present invention, when in IgGformat, have a high binding affinity for VEGF, i.e., have a Kd in therange of 1×10⁻⁸ M or less. Importantly, antibodies with such an affinityare in the established range that has been shown to be useful fortherapy. Preferably, the antibodies of the invention, when in IgGformat, have a binding affinity for VEGF (preferably human VEGF) thatcorresponds to a Kd of less than 20 nM, 15 nM or 10 nM, more preferablyof less than 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3,2.5, 2, 1.5 or 1 nM. For example, the binding affinity of the antibodiesof the invention, when in IgG format, may be 6.7×10⁻⁹ M or less, such asbeing about 7×10⁻⁹ M or about 6×10⁻⁹ M or as being 6.7×10⁻⁹ M. Anyappropriate method of determining Kd may be used. However, preferablythe Kd is determined by testing various concentrations of the testantibody against various concentrations of antigen (VEGF) in vitro toestablish a saturation curve, for example using the Lineweaver-Burkmethod, or preferably by using commercially available binding modelsoftware, such as the 1:1 binding model in the Biacore 3000 Evaluationsoftware. A suitable assay is described in Example 5 for illustrativepurposes. Preferably the Kd is determined by immobilizing antigen (VEGF)on a solid support, e.g. a Biacore chip, and assessing the binding ofthe antibody to the antigen. Preferably the binding affinity is assessedat room temperature, e.g. a temperature of 25° C., although it may alsobe assessed at other temperatures, e.g. 37° C. (e.g. body temperature).

As discussed elsewhere herein, preferred antibodies of the inventionbind to both human VEGF and murine VEGF. This is an important advantageto allow the most effective translation from preclinical studies toclinical use. For example, the ability of an antibody of the inventionto bind to both human VEGF and murine VEGF means that such antibodiescan be tested in preclinical studies using both syngeneic and xenografttumor models. Antibodies which do not bind to mouse VEGF cannot be usedin syngeneic mouse models.

In addition, the ability to bind both mouse and human VEGF means thatthe results shown by such antibodies of the invention in xenograft mousemodels are more likely to be representative of the activity of theantibodies in human subjects. The reason for this is that antibodieswhich can bind to human VEGF but not mouse VEGF (e.g. Avastin and 2C3)will bind to VEGF produced by the human tumor cells in the mouse modelbut will not be able to bind to endogenous murine VEGF. This is ofcourse unlike the situation in a human patient, in which VEGF producedby the tumor and endogenous VEGF would be present.

The potential disadvantage with such a situation is that an antibodywhich binds to human VEGF but not mouse VEGF might perform well in amouse xenograft model but this might not be reflected by a similarperformance in a human system where much more VEGF was present. In otherwords, the anti-tumor effect seen in a mouse xenograft system with anantibody which can bind to human VEGF but not mouse VEGF might lookbetter than the clinical reality. In contrast, if you are working withan antibody that can bind to both human and mouse VEGF then this willbind to all forms of VEGF present in the mouse model system and islikely to be more representative of the situation when the antibody isput into humans.

In the following descriptions of the compositions, immunoconjugates,pharmaceuticals, combinations, cocktails, kits, first and second medicaluses and all methods in accordance with this invention, the terms“antibody” and “immunoconjugate”, or an antigen-binding region orfragment thereof, unless otherwise specifically stated or made clearfrom the scientific terminology, refer to a range of VEGFR2-blocking,human anti-VEGF antibodies as well as to the specific r84 antibodies.

The terms “antibody” and “immunoglobulin”, as used herein, refer broadlyto any immunological binding agent or molecule that comprises a humanantigen binding domain, including polyclonal and monoclonal antibodies.Depending on the type of constant domain in the heavy chains, wholeantibodies are assigned to one of five major classes: IgA, IgD, IgE,IgG, and IgM. Several of these are further divided into subclasses orisotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. The heavy-chainconstant domains that correspond to the difference classes ofimmunoglobulins are termed α, δ, ε, γ and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

Generally, where whole antibodies rather than antigen binding regionsare used in the invention, IgG and/or IgM are preferred because they arethe most common antibodies in the physiological situation and becausethey are most easily made in a laboratory setting.

The “light chains” of mammalian antibodies are assigned to one of twoclearly distinct types: kappa (κ) and lambda (λ), based on the aminoacid sequences of their constant domains and some amino acids in theframework regions of their variable domains. There is essentially nopreference to the use of κ or λ light chain constant regions in theantibodies of the present invention.

As will be understood by those in the art, the immunological bindingreagents encompassed by the term “antibody” extend to all humanantibodies and antigen binding fragments thereof, including wholeantibodies, dimeric, trimeric and multimeric antibodies; bispecificantibodies; chimeric antibodies; recombinant and engineered antibodies,and fragments thereof.

The term “antibody” is thus used to refer to any human antibody-likemolecule that has an antigen binding region, and this term includesantibody fragments that comprise an antigen binding domain such as Fab′,Fab, F(ab′)₂, single domain antibodies (DABs), T and Abs dimer, Fv, scFv(single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies,diabodies, bispecific antibody fragments and the like.

The techniques for preparing and using various antibody-based constructsand fragments are well known in the art (see Kabat et al., 1991,specifically incorporated herein by reference). Diabodies, inparticular, are further described in EP 404, 097 and WO 93/11161;whereas linear antibodies are further described in Zapata et al. (1995).

Antibodies can be fragmented using conventional techniques. For example,F(ab′)₂ fragments can be generated by treating the antibody with pepsin.The resulting F(ab′)₂ fragment can be treated to reduce disulfidebridges to produce Fab′ fragments. Papain digestion can lead to theformation of Fab fragments. Fab, Fab′ and F(ab′)₂, scFv, Fv, dsFv, Fd,dAbs, T and Abs, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques or can be chemically synthesized. Techniques forproducing antibody fragments are well known and described in the art.For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; LeGall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young etal., 1995 further describe and enable the production of effectiveantibody fragments.

The human antibodies or antibody fragments can be produced naturally orcan be wholly or partially synthetically produced. Thus the antibody maybe from any appropriate source, for example recombinant sources and/orproduced in transgenic animals or transgenic plants, or in eggs usingthe IgY technology. Thus, the antibody molecules can be produced invitro or in vivo.

Preferably, the human antibody or antibody fragment comprises anantibody light chain variable region (V_(L)) that comprises three CDRdomains and an antibody heavy chain variable region (V_(H)) thatcomprises three CDR domains. Said VL and VH generally form the antigenbinding site.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region has a dimerof one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions (CDRs) of each variable domain interact to definean antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions (CDRs) conferantigen-binding specificity to the antibody.

However, it is well documented in the art that the presence of threeCDRs from the light chain variable domain and three CDRs from the heavychain variable domain of an antibody is not necessary for antigenbinding. Thus, constructs smaller than the above classical antibodyfragment are known to be effective.

For example, camelid antibodies (Hamers-Casterman et al., 1993; ArbabiGhahroudi et al., 1997) have an extensive antigen binding repertoire butare devoid of light chains. Also, results with single domain antibodiescomprising VH domains alone (Ward et al., 1989; Davies and Riechmann,1995) or VL domains alone (van den Beucken et al., 2001) show that thesedomains can bind to antigen with acceptably high affinities. Thus, threeCDRs can effectively bind antigen.

It is also known that a single CDR, or two CDRs, can effectively bindantigen. As a first example, a single CDR can be inserted into aheterologous protein and confer antigen binding ability on theheterologous protein, as exemplified by showing that a VH CDR3 regioninserted into a heterologous protein, such as GFP, confers antigenbinding ability on the heterologous protein (Kiss et al., 2006; Nicaiseet al., 2004).

It is further known that two CDRs can effectively bind antigen, and evenconfer superior properties than possessed by the parent antibody. Forexample, it has been shown (Qiu et al., 2007) that two CDRs from aparent antibody (a VH CDR1 and a VL CDR3 region) retain the antigenrecognition properties of the parent molecule but have a superiorcapacity to penetrate tumors. Joining these CDR domains with anappropriate linker sequence (e.g., from VH FR2) to orientate the CDRs ina manner resembling the native parent antibody produced even betterantigen recognition. Therefore, it is known in the art that it ispossible to construct antigen binding antibody mimetics comprising twoCDR domains (preferably one from a VH domain and one from a VL domain,more preferably, with one of the two CDR domains being a CDR3 domain)orientated by means of an appropriate framework region to maintain theconformation found in the parent antibody.

Thus, although preferred antibodies of the invention might comprise sixCDR regions (three from a light chain and three from a heavy chain),antibodies with fewer than six CDR regions and as few as one or two CDRregions are encompassed by the invention. In addition, antibodies withCDRs from only the heavy chain or light chain are also contemplated.

Preferred antibodies of the invention that bind to VEGF comprise atleast one heavy chain variable region that comprises three CDRs and atleast one light chain variable region that comprises three CDRs, whereinsaid light chain variable region comprises:

-   -   (a) a variable light (VL) CDR1 that has the amino acid sequence        of SEQ ID NO:8 or a sequence substantially homologous thereto,    -   (b) a VL CDR2 that has the amino acid sequence of SEQ ID NO:9 or        a sequence substantially homologous thereto, and    -   (c) a VL CDR3 that has the amino acid sequence of SEQ ID NO:10        or a sequence substantially homologous thereto.

Preferred heavy chain CDR regions for use in conjunction with thespecified light chain CDR regions are described elsewhere herein.However, other heavy chain variable regions that comprise three CDRs foruse in conjunction with the light chain variable regions of theinvention are also contemplated. Appropriate heavy chain variableregions which can be used in combination with the light chain variableregions of the invention and which give rise to an antibody which bindsVEGF can be readily identified by a person skilled in the art.

For example, a light chain variable region of the invention can becombined with a single heavy chain variable region or a repertoire ofheavy chain variable regions and the resulting antibodies tested forbinding to VEGF. It would be expected that a reasonable number of suchcombinations of light chain variable regions of the invention withdifferent heavy chain variable regions would retain the ability to bindVEGF. Indeed, this has been demonstrated with the preferred antibody ofthe invention (r84/PGN311) where it has been shown that the VL domain ofthis antibody can be combined with several different VH domains andstill retain the ability to bind VEGF. In these experiments 3 out of 7VH domains which were tested in combination with the VL domain of ther84 antibody showed significant binding to VEGF, which is a veryreasonable proportion and is evidence that the light chain variableregion of the antibodies of the invention is particularly important indetermining VEGF binding specificity and also that other heavy chainvariable regions which can be combined with the light chain variableregions of the invention and give rise to antibodies which bind VEGF canreadily be identified. Preferred heavy chain variable regions for use incombination with the light chain variable regions of the antibodies ofthe invention are those obtained or derived from antibodies or antibodyfragments which are known to bind to VEGF.

Similar methods could be used to identify alternative light chainvariable regions for use in combination with preferred heavy chainvariable regions of the invention.

In certain embodiments, the antibody or antibody fragment comprises allor a portion of a heavy chain constant region, such as an IgG1, IgG2,IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Preferably, theheavy chain constant region is an IgG1 heavy chain constant region, or aportion thereof. Furthermore, the antibody or antibody fragment cancomprise all or a portion of a kappa light chain constant region or alambda light chain constant region, or a portion thereof. All or part ofsuch constant regions may be produced naturally or may be wholly orpartially synthetic. Appropriate sequences for such constant regions arewell known and documented in the art. When a full complement of constantregions from the heavy and light chains are included in the antibodiesof the invention, such antibodies are typically referred to herein as“full length” antibodies or “whole” antibodies

Antibodies containing an Fc region are preferred for certain uses,particularly therapeutic uses in vivo, where the Fc region mediateseffector functions such as ADCC and CDC.

The term “substantially homologous” as used herein in connection with anamino acid or nucleic acid sequence includes sequences having at least70% or 75%, preferably at least 80%, and even more preferably at least85%, 90%, 95%, 96%, 97%, 98% or 99%, sequence identity to the amino acidor nucleic acid sequence disclosed. Substantially homologous sequencesof the invention thus include single or multiple base or amino acidalterations (additions, substitutions, insertions or deletions) to thesequences of the invention. At the amino acid level preferredsubstantially homologous sequences contain only 1, 2, 3, 4 or 5,preferably 1, 2 or 3, more preferably 1 or 2, altered amino acids, inone or more of the framework regions and/or one or more of the CDRsmaking up the sequences of the invention. Said alterations can be withconservative or non-conservative amino acids. Preferably saidalterations are conservative amino acid substitutions.

The substantially homologous nucleic acid sequences also includenucleotide sequences that hybridize to the nucleic acid sequencesdisclosed (or their complementary sequences), e.g., hybridize tonucleotide sequences encoding one or more of the light chain or heavychain CDRs of the invention, the light or heavy chain variable regionsof the invention, or the antibodies of the invention (or hybridize totheir complementary sequences), under at least moderately stringenthybridization conditions.

The term “substantially homologous” also includes modifications orchemical equivalents of the amino acid and nucleotide sequences of thepresent invention that perform substantially the same function as theproteins or nucleic acid molecules of the invention in substantially thesame way. For example, any substantially homologous antibody (or thesubstantially homologous nucleic acid encoding it) should retain theability to bind to VEGF as described above. Preferably, anysubstantially homologous binding protein should retain the ability tospecifically bind to the same epitope of VEGF as recognized by thebinding protein in question, for example, the same epitope recognized bythe CDR domains of the invention or the VH and VL domains of theinvention as described herein. Binding to the same epitope/antigen canbe readily tested by methods well known and described in the art, e.g.,using binding assays, e.g., a competition assay.

Thus, a person skilled in the art will appreciate that binding assayscan be used to test whether “substantially homologous” antibodies havethe same binding specificities as the antibodies and antibody fragmentsof the invention, for example, binding assays such as ELISA assays orBiacore assays can readily be used to establish whether such“substantially homologous” antibodies can bind to VEGF. As outlinedbelow, a competition binding assay can be used to test whether“substantially homologous” antibodies retain the ability to specificallybind to substantially the same epitope of VEGF as recognized by theantibodies of the invention. The method described below is only oneexample of a suitable competition assay. The skilled person will beaware of other suitable methods and variations.

An exemplary competition assay involves assessing the binding of variouseffective concentrations of an antibody of the invention to VEGF in thepresence of varying concentrations of a test antibody (e.g., asubstantially homologous antibody). The amount of inhibition of bindinginduced by the test antibody can then be assessed. A test antibody thatshows increased competition with an antibody of the invention atincreasing concentrations (i.e., increasing concentrations of the testantibody result in a corresponding reduction in the amount of antibodyof the invention binding to VEGF) is evidence of binding tosubstantially the same epitope. Preferably, the test antibodysignificantly reduces the amount of antibody of the invention that bindsto VEGF. Preferably, the test antibody reduces the amount of antibody ofthe invention that binds to VEGF by at least about 80%. ELISA assays areappropriate for assessing inhibition of binding in such a competitionassay but other suitable techniques would be well known to a personskilled in the art.

Substantially homologous sequences of proteins of the invention include,without limitation, conservative amino acid substitutions, or forexample alterations that do not effect the VH, VL or CDR domains of theantibodies, e.g., include scFv antibodies where a different linkersequence is used or antibodies where tag sequences or other componentsare added that do not contribute to the binding of antigen, oralterations to convert one type or format of antibody molecule orfragment to another type or format of antibody molecule or fragment(e.g., conversion from Fab to scFv or vice versa), or the conversion ofan antibody molecule to a particular class or subclass of antibodymolecule (e.g., the conversion of an antibody molecule to IgG or asubclass thereof, e.g., IgG1 or IgG3).

A “conservative amino acid substitution”, as used herein, is one inwhich the amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Homology may be assessed by any convenient method. However, fordetermining the degree of homology between sequences, computer programsthat make multiple alignments of sequences are useful, for instanceClustal W (Thompson et al., 1994). If desired, the Clustal W algorithmcan be used together with BLOSUM 62 scoring matrix (Henikoff andHenikoff, 1992) and a gap opening penalty of 10 and gap extensionpenalty of 0.1, so that the highest order match is obtained between twosequences wherein at least 50% of the total length of one of thesequences is involved in the alignment. Other methods that may be usedto align sequences are the alignment method of Needleman and Wunsch(1970), as revised by Smith and Waterman (1981) so that the highestorder match is obtained between the two sequences and the number ofidentical amino acids is determined between the two sequences. Othermethods to calculate the percentage identity between two amino acidsequences are generally art recognized and include, for example, thosedescribed by Carillo and Lipton (1988) and those described inComputational Molecular Biology, Lesk, e.d. Oxford University Press, NewYork, 1988, Biocomputing: Informatics and Genomics Projects.

Generally, computer programs will be employed for such calculations.Programs that compare and align pairs of sequences, like ALIGN (Myersand Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990) andgapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereuxet al., 1984) are also useful for this purpose. Furthermore, the Daliserver at the European Bioinformatics institute offers structure-basedalignments of protein sequences (Holm, 1993; 1995; 1998).

By way of providing a reference point, sequences according to thepresent invention having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% homology, sequence identity etc. may be determined using the ALIGNprogram with default parameters (for instance available on Internet atthe GENESTREAM network server, IGH, Montpellier, France).

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected that promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g., 20,25, 30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600/1), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule, a1% mismatch may be assumed to result in about a 1° C. decrease in Tm.For example, if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm-5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C.

By way of further example, sequences that “hybridize” are thosesequences binding (hybridizing) under non-stringent conditions (e.g.,6×SSC, 50% formamide at room temperature) and washed under conditions oflow stringency (e.g., 2×SSC, room temperature, more preferably 2×SSC,42° C.) or conditions of higher stringency (e.g., 2×SSC, 65° C.) (whereSSC=0.15M NaCl, 0.015M sodium citrate, pH 7.2).

It is understood, however, that equivalent stringencies may be achievedusing alternative buffers, salts and temperatures. Additional guidanceregarding hybridization conditions may be found in: Current Protocols inMolecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6 and in:Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Laboratory Press, 1989, Vol. 3.

Generally speaking, sequences that hybridize under conditions of highstringency are preferred, as are sequences which, but for the degeneracyof the code, would hybridize under high stringency conditions.

In other preferred embodiments, second generation antibodies areprovided that have enhanced or superior properties in comparison to anoriginal VEGFR2-blocking, human anti-VEGF antibody, such as r84. Forexample, the second generation antibodies may have a stronger bindingaffinity, more effective blocking of VEGF binding to VEGFR2, morespecific blocking of VEGF binding to VEGFR2, even less blocking of VEGFbinding to VEGFR1, enhanced ability to inhibit VEGF-inducedproliferation and/or migration of endothelial cells, superior ability toinhibit VEGF-induced vascular permeability, and preferably, an increasedability to inhibit VEGF-induced angiogenesis in vivo, and to treatangiogenic diseases, including vascularized tumors.

Comparisons to identify effective second generation antibodies arereadily conducted and quantified, e.g., using one or more of the variousassays described in detail herein. Second generation antibodies thathave an enhanced biological property or activity of at least about2-fold, 5-fold, 10-fold, 20-fold, and preferably, at least about50-fold, in comparison to the VEGFR2-blocking, human anti-VEGFantibodies of the present invention, as exemplified by the r84 antibody,are encompassed by the present invention.

The antibody, binding protein and nucleic acid molecules of theinvention are generally “isolated” or “purified” molecules insofar asthey are distinguished from any such components that may be present insitu within a human or animal body or a tissue sample derived from ahuman or animal body. The sequences may, however, correspond to or besubstantially homologous to sequences as found in a human or animalbody. Thus, the term “isolated” or “purified” as used herein inreference to nucleic acid molecules or sequences and proteins orpolypeptides, e.g., antibodies, refers to such molecules when isolatedfrom, purified from, or substantially free of their natural environment,e.g., isolated from or purified from the human or animal body (if indeedthey occur naturally), or refers to such molecules when produced by atechnical process, i.e., includes recombinant and synthetically producedmolecules.

Thus, when used in connection with a nucleic acid molecule, such termsmay refer to a nucleic acid substantially free of material with which itis naturally associated such as other nucleic acids/genes orpolypeptides. These terms may also refer to a nucleic acid substantiallyfree of cellular material or culture medium when produced by recombinantDNA techniques, or substantially free of chemical precursors, or otherchemicals when chemically synthesized. An isolated or purified nucleicacid may also be substantially free of sequences that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) from which the nucleic acid is derived or sequences thathave been made to flank the nucleic acid (e.g., tag sequences or othersequence that have no therapeutic value) by, for example, geneticengineering.

Thus, when used in connection with a protein or polypeptide moleculesuch as light chain CDRs 1, 2 and 3, heavy chain CDRs 1, 2 and 3, lightchain variable regions, heavy chain variable regions, and bindingproteins or antibodies of the invention, including full lengthantibodies, the term “isolated” or “purified” typically refers to aprotein substantially free of cellular material or other proteins fromthe source from which it is derived. In some embodiments, particularlywhere the protein is to be administered to humans or animals, suchisolated or purified proteins are substantially free of culture mediumwhen produced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. Such isolated or purifiedproteins may also be free of flanking sequences such as those describedabove for the isolated nucleic acid molecules.

The term “nucleic acid sequence” or “nucleic acid molecule” as usedherein refers to a sequence of nucleoside or nucleotide monomerscomposed of naturally occurring bases, sugars and intersugar (backbone)linkages. The term also includes modified or substituted sequencescomprising non-naturally occurring monomers or portions thereof. Thenucleic acid sequences of the present invention may be deoxyribonucleicacid sequences (DNA) or ribonucleic acid sequences (RNA) and may includenaturally occurring bases including adenine, guanine, cytosine,thymidine and uracil. The sequences may also contain modified bases.Examples of such modified bases include aza and deaza adenine, guanine,cytosine, thymidine and uracil; and xanthine and hypoxanthine. Thenucleic acid molecules may be double stranded or single stranded. Thenucleic acid molecules may be wholly or partially synthetic orrecombinant.

The term “human” as used herein in connection with antibody moleculesand binding proteins first refers to antibodies and binding proteinshaving variable regions (e.g., V_(H), V_(L), CDR or FR regions) and,optionally, constant antibody regions, isolated or derived from a humanrepertoire or derived from or corresponding to sequences found inhumans, e.g., in the human germline or somatic cells. The r84 antibodyis an example of such a human antibody molecule wherein the variableregions have been isolated from a human repertoire.

The “human” antibodies and binding proteins of the invention furtherinclude amino acid residues not encoded by human sequences, e.g.,mutations introduced by random or site directed mutations in vitro, forexample mutations introduced by in vitro cloning or PCR. Particularexamples of such mutations are mutations that involve conservativesubstitutions or other mutations in a small number of residues of theantibody or binding protein, e.g., in 5, 4, 3, 2 or 1 of the residues ofthe antibody or binding protein, preferably e.g., in 5, 4, 3, 2 or 1 ofthe residues making up one or more of the CDRs of the antibody orbinding protein. Certain examples of such “human” antibodies includeantibodies and variable regions that have been subjected to standardmodification techniques to reduce the amount of potentially immunogenicsites.

Thus, the “human” antibodies of the invention include sequences derivedfrom and related to sequences found in humans, but which may notnaturally exist within the human antibody germline repertoire in vivo.In addition, the human antibodies and binding proteins of the presentinvention include proteins comprising human consensus sequencesidentified from human sequences, or sequences substantially homologousto human sequences.

In addition, the human antibodies and binding proteins of the presentinvention are not limited to combinations of V_(H), V_(L), CDR or FRregions that are themselves found in combination in human antibodymolecules. Thus, the human antibodies and binding proteins of theinvention can include or correspond to combinations of such regions thatdo not necessarily exist naturally in humans.

In preferred embodiments, the human antibodies will be fully humanantibodies. “Fully human” antibodies, as used herein, are antibodiescomprising “human” variable region domains and/or CDRs, as definedabove, without substantial non-human antibody sequences or without anynon-human antibody sequences. For example, antibodies comprising humanvariable region domains and/or CDRs “without substantial non-humanantibody sequences” are antibodies, domains and/or CDRs in which onlyabout 5, 4, 3, 2 or 1 amino acids are amino acids that are not encodedby human antibody sequences. Thus, “fully human” antibodies aredistinguished from “humanized” antibodies, which are based onsubstantially non-human variable region domains, e.g., mouse variableregion domains, in which certain amino acids have been changed to bettercorrespond with the amino acids typically present in human antibodies.

The “fully human” antibodies of the invention may be human variableregion domains and/or CDRs without any other substantial antibodysequences, such as being single chain antibodies. Alternatively, the“fully human” antibodies of the invention may be human variable regiondomains and/or CDRs integral with or operatively attached to one or morehuman antibody constant regions. Certain preferred fully humanantibodies are IgG antibodies with the full complement of IgG constantregions.

In other embodiments, “human” antibodies of the invention will bepart-human chimeric antibodies. “Part-human chimeric” antibodies, asused herein, are antibodies comprising “human” variable region domainsand/or CDRs operatively attached to, or grafted onto, a constant regionof a non-human species, such as rat or mouse. Such part-human chimericantibodies may be used, for example, in pre-clinical studies, whereinthe constant region will preferably be of the same species of animalused in the pre-clinical testing. These part-human chimeric antibodiesmay also be used, for example, in ex vivo diagnostics, wherein theconstant region of the non-human species may provide additional optionsfor antibody detection.

The term “fragment” as used herein refers to fragments of biologicalrelevance, e.g., fragments that contribute to antigen binding, e.g.,form part of the antigen binding site, and/or contribute to theinhibition or reduction in function of the VEGF antigen and/orcontribute to the prevention of the VEGF antigen interacting with thenatural ligand, VEGFR2. Certain preferred fragments comprise a heavychain variable region (V_(H) domain) and/or a light chain variableregion (V_(L) domain) of the antibodies of the invention. Otherpreferred fragments comprise one or more of the heavy chain CDRs of theantibodies of the invention (or of the V_(H) domains of the invention),or one or more of the light chain CDRs of the antibodies of theinvention (or of the V_(L) domains of the invention). Certain preferredfragments are at least 5 amino acids in length and comprise at least oneCDR region, preferably a CDR3 region, more preferably a heavy chain CDR3region.

In embodiments where the antibodies of the invention comprise a fragmentof any of the defined sequences (for example comprise a fragment of SEQID NO:21, e.g., are antibodies comprising V_(H) and/or V_(L) domains ofthe invention, or are antibodies or binding proteins comprising one ormore CDRs of the invention, then these regions/domains are generallyseparated within the antibody or binding protein so that eachregion/domain can perform its biological function and so that thecontribution to antigen binding is retained. Thus, the V_(H) and V_(L)domains are preferably separated by appropriate scaffoldsequences/linker sequences and the CDRs are preferably separated byappropriate framework regions such as those found in naturally occurringantibodies and/or effective engineered antibodies. Thus, the V_(H),V_(L) and individual CDR sequences of the invention are preferablyprovided within or incorporated into an appropriate framework orscaffold to enable antigen binding. Such framework sequences or regionsmay correspond to naturally occurring framework regions, FR1, FR2, FR3and/or FR4, as appropriate to form an appropriate scaffold, or maycorrespond to consensus framework regions, for example identified bycomparing various naturally occurring framework regions. Alternatively,non-antibody scaffolds or frameworks, e.g., T cell receptor frameworkscan be used.

Appropriate sequences that can be used for framework regions are wellknown and documented in the art and any of these may be used. Preferredsequences for framework regions are one or more of the framework regionsmaking up the V_(H) and/or V_(L) domains of the invention, i.e., one ormore of the framework regions disclosed in SEQ ID NO:21 or in Table 1,or framework regions substantially homologous thereto, and in particularframework regions that allow the maintenance of antigen specificity, forexample framework regions that result in substantially the same or thesame 3D structure of the antibody. In certain preferred embodiments, allfour of the variable light chain (SEQ ID NOs:15, 16, 17 and 18) and/orvariable heavy chain (SEQ ID NOs:11, 12, 13 and 14), as appropriate, FRregions of SEQ ID NO:21 (also shown in Table 1), or FR regionssubstantially homologous thereto, are found in the antibodies of theinvention.

In addition, although preferred antibodies of the invention are made upof V_(H), V_(L) or CDRs of the invention, it should be noted that theantibodies of the invention also encompass one or more V_(H), V_(L) orCDRs of the invention in combination with other V_(H), V_(L) or CDRs notof the invention, provided that the VEGF binding properties of theantibodies or binding proteins of the invention as outlined above arestill present.

The term “heavy chain complementarity determining region” (“heavy chainCDR”) as used herein refers to regions of hypervariability within theheavy chain variable region (V_(H) domain) of an antibody molecule. Theheavy chain variable region has three CDRs termed heavy chain CDR1,heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxyterminus. The heavy chain variable region also has four frameworkregions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxyterminus). These framework regions separate the CDRs.

The term “heavy chain variable region” (V_(H) domain) as used hereinrefers to the variable region of a heavy chain of an antibody molecule.

The term “light chain complementarity determining region” (“light chainCDR”) as used herein refers to regions of hypervariability within thelight chain variable region (V_(L) domain) of an antibody molecule.Light chain variable regions have three CDRs termed light chain CDR1,light chain CDR2 and light chain CDR3 from the amino terminus to thecarboxy terminus. The light chain variable region also has fourframework regions (FR1, FR2, FR3 and FR4 from the amino terminus tocarboxy terminus). These framework regions separate the CDRs.

The term “light chain variable region” (V_(L) domain) as used hereinrefers to the variable region of a light chain of an antibody molecule.

It should be noted that the Kabat nomenclature is followed herein, wherenecessary, in order to define the positioning of the CDRs (Kabat et al.,1991, specifically incorporated herein by reference).

A person skilled in the art will appreciate that the proteins andpolypeptides of the invention, such as the light and heavy CDRs, thelight and heavy chain variable regions, antibodies, antibody fragments,and immunoconjugates, may be prepared in any of several ways well knownand described in the art, but are most preferably prepared usingrecombinant methods.

Nucleic acid fragments encoding the light and heavy chain variableregions of the antibodies of the invention can be derived or produced byany appropriate method, e.g., by cloning or synthesis. Such sequencescould, for example, be prepared by cloning appropriate sequences frome.g., human germ line genes and then making any necessary modificationsto the germ line sequences to obtain the sequences of the inventionusing methods well known and described in the art. An alternative andmore efficient method would be to synthesize the appropriate light orheavy chain variable region sequence as overlapping primers, and useprimer extension to obtain the full sequence. This full sequence couldthen be amplified via PCR with primers containing appropriaterestriction sites for further cloning and manipulation, e.g., forcloning into an appropriate expression vector. Five to seven overlappingprimers per variable region are normally be sufficient, thereby makingthis technique very efficient and precise.

Once nucleic acid fragments encoding the light and heavy chain variableregions of the antibodies of the invention have been obtained, thesefragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region fragments intofull length antibody molecules with appropriate constant region domains,or into particular formats of antibody fragment discussed elsewhereherein, e.g., Fab fragments, scFv fragments, etc. Typically, or as partof this further manipulation procedure, the nucleic acid fragmentsencoding the antibody molecules of the invention are generallyincorporated into an appropriate expression vector in order tofacilitate production of the antibodies of the invention.

Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses), so long as the vector iscompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule of the invention and regulatorysequences selected on the basis of the host cells to be used forexpression, which are operatively linked to the nucleic acid molecule.Operatively linked is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner that allows expression of thenucleic acid.

The invention therefore contemplates a recombinant expression vectorcontaining a nucleic acid molecule of the invention, or a fragmentthereof, and the necessary regulatory sequences for the transcriptionand translation of the protein sequence encoded by the nucleic acidmolecule of the invention.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, 1990).Selection of appropriate regulatory sequences is dependent on the hostcell chosen as discussed below, and may be readily accomplished by oneof ordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

The recombinant expression vectors of the invention may also contain aselectable marker gene that facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asneomycin and hygromycin that confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. Transcription of the selectable markergene is monitored by changes in the concentration of the selectablemarker protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. If the selectable marker geneencodes a protein conferring antibiotic resistance such as neomycinresistance transformant cells can be selected with G418. Cells that haveincorporated the selectable marker gene will survive, while the othercells die. This makes it possible to visualize and assay for expressionof recombinant expression vectors of the invention and in particular todetermine the effect of a mutation on expression and phenotype. It willbe appreciated that selectable markers can be introduced on a separatevector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes that encode afusion moiety that provides increased expression of the recombinantprotein; increased solubility of the recombinant protein; and aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification (for example appropriate “tags” to enablepurification and/or identification may be present, e.g., His tags or myctags). For example, a proteolytic cleavage site may be added to thetarget recombinant protein to allow separation of the recombinantprotein from the fusion moiety subsequent to purification of the fusionprotein. Typical fusion expression vectors include pGEX (Amrad Corp.,Melbourne, Australia), pMal (New England Biolabs, Beverly, Mass.) andpRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to therecombinant protein.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” and “transfection” are intended toencompass introduction of nucleic acid (e.g., a vector) into a cell byone of many possible techniques known in the art. The term “transformedhost cell” as used herein is intended to also include cells capable ofglycosylation that have been transformed with a recombinant expressionvector of the invention. Prokaryotic cells can be transformed withnucleic acid by, for example, electroporation or calcium-chloridemediated transformation. For example, nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al., 1989, and other laboratory textbooks.

Suitable host cells include a wide variety of eukaryotic host cells andprokaryotic cells. For example, the proteins of the invention may beexpressed in yeast cells or mammalian cells. Other suitable host cellscan be found in Goeddel, 1990. In addition, the proteins of theinvention may be expressed in prokaryotic cells, such as Escherichiacoli (Zhang et al., 2004).

Yeast and fungi host cells suitable for carrying out the presentinvention include, but are not limited to Saccharomyces cerevisiae, thegenera Pichia or Kluyveromyces and various species of the genusAspergillus. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari. et al., 1987), pMFa (Kurjan and Herskowitz,1982), pJRY88 (Schultz et al., 1987), and pYES2 (Invitrogen Corporation,San Diego, Calif.). Protocols for the transformation of yeast and fungiare well known to those of ordinary skill in the art (see Hinnen et al.,1978; Ito et al., 1983, and Cullen et al. 1987).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573) and NS-1 cells. Suitable expression vectors for directingexpression in mammalian cells generally include a promoter (e.g.,derived from viral material such as polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40), as well as other transcriptionaland translational control sequences. Examples of mammalian expressionvectors include pCDM8 (Seed, B., 1987) and pMT2PC (Kaufman et al.,1987).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, the proteins of the invention may be expressedfrom plant cells (see Sinkar et al., 1987, which reviews the use ofAgrobacterium rhizogenes vectors; see also Zambryski et al., 1984, whichdescribes the use of expression vectors for plant cells, including,among others, PAPS2022, PAPS2023, and PAPS2034).

Insect cells suitable for carrying out the present invention includecells and cell lines from Bombyx, Trichoplusia or Spodotera species.Baculovirus vectors available for expression of proteins in culturedinsect cells (SF 9 cells) include the pAc series (Smith et al., 1983)and the pVL series (Luckow and Summers 1989). Some baculovirus-insectcell expression systems suitable for expression of the recombinantproteins of the invention are described in PCT/US/02442.

Alternatively, the proteins of the invention may also be expressed innon-human transgenic animals such as, rats, rabbits, sheep and pigs(Hammer et al. 1985; Palmiter et al. 1983; Brinster et al. 1985;Palmiter and Brinster 1985, and U.S. Pat. No. 4,736,866).

The proteins of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield (1964); Frische et al., 1996) or synthesisin homogenous solution (Houbenweyl, 1987).

N-terminal or C-terminal fusion proteins comprising the antibodies andproteins of the invention conjugated to other molecules, such asproteins, may be prepared by fusing through recombinant techniques. Theresultant fusion proteins contain an antibody or protein of theinvention fused to the selected protein or marker protein, or tagprotein as described herein. The antibodies and proteins of theinvention may also be conjugated to other proteins by known techniques.For example, the proteins may be coupled using heterobifunctionalthiol-containing linkers as described in WO 90/10457,N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5thioacetate. Examples of proteins that may be used to prepare fusionproteins or conjugates include cell binding proteins such asimmunoglobulins, hormones, growth factors, lectins, insulin, low densitylipoprotein, glucagon, endorphins, transferrin, bombesin,asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA),and truncated myc.

Irrespective of the manner of preparation of a first VEGFR2-blocking,anti-VEGF antibody nucleic acid segment, further suitable antibodynucleic acid segments may be readily prepared by standard molecularbiological techniques. In order to confirm that any variant, mutant orsecond generation VEGFR2-blocking, anti-VEGF antibody nucleic acidsegment is suitable for use in the present invention, the nucleic acidsegment will be tested to confirm expression of a VEGFR2-blocking,anti-VEGF antibody in accordance with the present invention. Preferably,the variant, mutant or second generation nucleic acid segment will alsobe tested to confirm hybridization under standard, more preferably,standard stringent hybridization conditions. Exemplary suitablehybridization conditions include hybridization in about 7% sodiumdodecyl sulfate (SDS), about 0.5 M NaPO₄, about 1 mM EDTA at about 50°C.; and washing with about 1% SDS at about 42° C.

As a variety of human antibodies may be readily prepared, the treatmentmethods of the invention may be executed by providing to the animal orpatient at least a first nucleic acid segment or molecule that expressesa biologically effective amount of at least a first VEGFR2-blocking,human anti-VEGF antibody of the invention in the patient. The “nucleicacid segment or molecule that expresses a VEGFR2-blocking, humananti-VEGF antibody” will generally be in the form of at least anexpression construct or vector, and may be in the form of an expressionconstruct or vector comprised within a virus or within a recombinanthost cell. Preferred gene therapy vectors of the present invention willgenerally be viral vectors, such as comprised within a recombinantretrovirus, herpes simplex virus (HSV), adenovirus, adeno-associatedvirus (AAV), cytomegalovirus (CMV), and the like.

Thus, this invention further provides nucleic acid segments or moleculescomprising nucleotide sequences that encode the antibodies of thepresent invention. Nucleic acid molecules substantially homologous tosuch sequences are also included. Preferred nucleic acid moleculesencode the amino acid sequence set out in SEQ ID NO:21. More preferrednucleic acid molecules comprise the nucleic acid sequence as defined inSEQ ID NO:20 or a sequence substantially homologous thereto.

A yet further aspect provides an expression construct or expressionvector comprising one or more of the nucleic acid segments or moleculesof the invention. Preferably the expression constructs or vectors arerecombinant. Preferably said constructs or vectors further comprise thenecessary regulatory sequences for the transcription and translation ofthe protein sequence encoded by the nucleic acid molecule of theinvention.

A yet further aspect provides a host cell or virus comprising one ormore expression constructs or expression vectors of the invention. Alsoprovided are host cells or viruses comprising one or more of the nucleicacid molecules of the invention. A host cell or virus expressing anantibody of the invention forms a yet further aspect.

A yet further aspect of the invention provides a method of producing anantibody of the present invention comprising a step of culturing thehost cells of the invention. Preferred methods comprise the steps of (i)culturing a host cell comprising one or more of the recombinantexpression vectors or one or more of the nucleic acid sequences of theinvention under conditions suitable for the expression of the encodedantibody or protein; and optionally (ii) isolating the antibody orprotein from the host cell or from the growth medium/supernatant. Suchmethods of production may also comprise a step of purification of theantibody or protein product and/or formulating the antibody or productinto a composition including at least one additional component, such asa pharmaceutically acceptable carrier or excipient.

In embodiments when the antibody or protein of the invention is made upof more than one polypeptide chain (e.g., certain fragments such as Fabfragments), then all the polypeptides are preferably expressed in thehost cell, either from the same or a different expression vector, sothat the complete proteins, e.g., binding proteins of the invention, canassemble in the host cell and be isolated or purified therefrom.

The antibodies of the invention may also be used to produce furtherantibodies that bind to VEGF. Such uses involve for example theaddition, deletion, substitution or insertion of one or more amino acidsin the amino acid sequence of a parent antibody to form a new antibody,wherein said parent antibody is one of the antibodies of the inventionas defined elsewhere herein, and testing the resulting new antibody toidentify antibodies specific for VEGF. Such methods can be used to formmultiple new antibodies that can all be tested for their ability to bindVEGF. Preferably said addition, deletion, substitution or insertion ofone or more amino acids takes place in one or more of the CDR domains.

Such modification or mutation to a parent antibody can be carried out inany appropriate manner using techniques well known and documented in theart, for example by carrying out methods of random or directedmutagenesis. If directed mutagenesis is to be used then one strategy toidentify appropriate residues for mutagenesis utilizes the resolution ofthe crystal structure of the binding protein-antigen complex, e.g., theAb-Ag complex, to identify the key residues involved in the antigenbinding (Davies and Cohen, 1996). Subsequently, those residues can bemutated to enhance the interaction. Alternatively, one or more aminoacid residues can simply be targeted for directed mutagenesis and theeffect on binding to tumor cells assessed.

Random mutagenesis can be carried out in any appropriate way, e.g., byerror-prone PCR, chain shuffling or mutator E. coli strains.

Thus, one or more of the V_(H) domains of the invention can be combinedwith a single V_(L) domain or a repertoire of V_(L) domains from anyappropriate source and the resulting new antibodies tested to identifyantibodies specific for VEGF. Conversely, one or more of the V_(L)domains of the invention can be combined with a single V_(H) domain orrepertoire of V_(H) domains from any appropriate source and theresulting new antibodies tested to identify antibodies specific forVEGF. For example, as discussed above, it has been shown that the V_(L)domain of the preferred antibody of the invention (r84/PGN311) can becombined with several different V_(H) domains and still retain theability to bind VEGF.

Similarly, one or more, or preferably all three CDRs of the V_(H) and/orV_(L) domains of the invention can be grafted into a single V_(H) and/orV_(L) domain or a repertoire of V_(H) and/or V_(L) domains, asappropriate, and the resulting new antibodies tested to identifyantibodies specific for VEGF.

The targeted mutations of the CDRs, especially CDR3 of the light and/orheavy chains, have been shown to be an effective technique forincreasing antibody affinity and are preferred. Preferably, blocks of 3to 4 amino acids of the CDR3 or specific regions called “hot-spots” aretargeted for mutagenesis.

“Hot spots” are the sequences where somatic hypermutation takes place invivo (Neuberger and Milstein, 1995). The hotspot sequences can bedefined as consensus nucleotide sequences in certain codons. Theconsensus sequence is the tetranucleotide, RGYW, in which R can beeither A or G, Y can be C or T and W can be either A or T (Neuberger andMilstein, 1995). In addition, the serine residues encoded by thenucleotides AGY are predominantly present in the CDRs regions of thevariable domain over those encoded by TCN corresponding to a potentialhot-spot sequences (Wagner et al., 1995).

Thus, the nucleotide sequence of the CDRs of the heavy and light chainsof each antibody of the invention can be scanned for the presence of thehot-spot sequences and AGY codons. The identified hot-spots of the CDRregions of the light and heavy chain can then optionally be compared tothe germinal sequences of the heavy and light chains using theInternational ImMunoGen Tics database (IMGT,http://imgt.cines.fr/textes/vquest/) (Davies et al., 1990). A sequence,identical to the germ line, suggest that somatic mutation has notoccurred; therefore random mutations can be introduced mimicking thesomatic events occurring in vivo or alternatively, site directedmutagenesis can be carried out, e.g., at the hot spots and/or AGYcodons. In contrast, a different sequence shows that some somaticmutations have already occurred. It will remain to be determined if thein vivo somatic mutation was optimal.

Preferred hot-spots for mutation are those that code for exposed aminoacids and preferably those that encode amino acids that form part of theantigen binding sites. Other preferred hot-spots for mutation are thosethat code for non-conserved amino acids. The hot-spots that code forburied or conserved amino acids within the CDRs are preferably notmutagenized. These residues are usually critical for the overallstructure and are unlikely to interact with the antigen since they areburied.

Methods of carrying out the above described manipulation of amino acidsand protein domains are well known to a person skilled in the art. Forexample, said manipulations could conveniently be carried out by geneticengineering at the nucleic acid level wherein nucleic acid moleculesencoding appropriate binding proteins and domains thereof are modifiedsuch that the amino acid sequence of the resulting expressed protein isin turn modified in the appropriate way.

Testing the ability of one or more new antibodies to specifically bindto VEGF can be carried out by any appropriate method, which are wellknown and described in the art. VEGF samples are widely available (seethe Examples) and these can readily be used to assay binding, forexample by conventional methods such as ELISA, affinity chromatography,etc.

The new antibodies produced by these methods will preferably have ahigher or enhanced affinity (or at least an equivalent affinity) forVEGF as the parent antibodies and can be treated and used in the sameway as the antibodies of the invention as described elsewhere herein(e.g., for therapy, diagnosis, in compositions etc).

New antibodies produced, obtained or obtainable by these methods form ayet further aspect of the invention.

This invention further provides compositions comprising at least onehuman antibody or antibody fragment of the invention, optionallyincluding a diluent. Such compositions may be pharmaceuticallyacceptable compositions or compositions for use in laboratory studies.In terms of the pharmaceutical compositions, they may preferably beformulated for parenteral administration, such as for intravenousadministration, or for ocular administration.

The present invention provides a number of methods and uses of the humanantibodies and antibody fragments of the invention. Concerning allmethods, the terms “a” and “an” are used to mean “at least one”, “atleast a first”, “one or more” or “a plurality” of steps in the recitedmethods, except where specifically stated. This is particularly relevantto the administration steps in the treatment methods. Thus, not only maydifferent doses be employed with the present invention, but differentnumbers of doses, e.g., injections, may be used, up to and includingmultiple injections. Combined therapeutics may be used, administeredbefore, after or during administration of the anti-VEGF therapeuticantibody.

Various useful in vitro methods and uses of the antibodies of theinvention are provided that have important biological implications.First provided are methods of, and uses in, binding VEGF, whichgenerally comprise effectively contacting a composition comprising VEGF,preferably free (non-receptor bound) VEGF with at least a firstVEGFR2-blocking, anti-VEGF antibody of the invention, or antigen-bindingfragment thereof.

Methods of, and uses in, detecting VEGF are provided, which generallycomprise contacting a composition suspected of containing VEGF with atleast a first human antibody of the invention, or antigen-bindingfragment thereof, under conditions effective to allow the formation ofVEGF/antibody complexes and detecting the complexes so formed. Thedetection methods and uses may be used in connection with biologicalsamples, e.g., in diagnostics for angiogenesis and tumors, anddiagnostic kits based thereon are also provided.

The present invention provides methods of, and uses in, preferentiallyor specifically inhibiting VEGF binding to the VEGF receptor VEGFR2,which generally comprise contacting, in the presence of VEGF, apopulation of cells or tissues that includes endothelial cells thatexpress VEGFR2 (KDR/Flk-1) with a composition comprising a biologicallyeffective amount of at least a first VEGFR2-blocking, human anti-VEGFantibody of the invention, or an antigen-binding fragment thereof, underconditions effective to inhibit VEGF binding to the VEGF receptorVEGFR2.

Methods of, and uses in, significantly inhibiting VEGF binding to theVEGF receptor VEGFR2, without significantly inhibiting VEGF binding tothe VEGF receptor VEGFR1 are provided. These methods comprisecontacting, in the presence of VEGF, a population of cells or tissuesthat includes a population of endothelial cells that express VEGFR2(KDR/Flk-1) and VEGFR1 (Flt-1) with a composition comprising abiologically effective amount of at least a first VEGFR2-blocking, humananti-VEGF antibody of the invention, or an antigen-binding fragmentthereof, under conditions effective to inhibit VEGF binding to the VEGFreceptor VEGFR2, without significantly inhibiting VEGF binding to theVEGF receptor VEGFR1.

Further methods and uses of the invention are in analyzing thebiological roles of the VEGF receptors termed VEGFR2 and VEGFR1,comprising the steps of:

-   -   (a) contacting a biological composition or tissue that comprises        VEGF and a population of cells that express VEGFR2 (KDR/Flk-1)        and VEGFR1 (Flt-1) receptors with a composition comprising a        biologically effective amount of at least a first        VEGFR2-blocking, human anti-VEGF antibody of the invention, or        an antigen-binding fragment thereof; and    -   (b) determining the effect of the VEGFR2-blocking, anti-VEGF        antibody of the invention on at least a first biological        response to VEGF; wherein:        -   (i) an alteration in a biological response in the presence            of the VEGFR2-blocking, anti-VEGF antibody of the invention            is indicative of a response mediated by the VEGFR2 receptor;            and        -   (ii) the maintenance of a biological response in the            presence of the VEGFR2-blocking, anti-VEGF antibody of the            invention is indicative of a response mediated by the VEGFR1            receptor.

Proliferation inhibition methods and uses are provided, including thoseto specifically inhibit VEGF-induced endothelial cell proliferationand/or migration, which generally comprise contacting a population ofcells or tissues that includes a population of endothelial cells andVEGF with a composition comprising a biologically effective amount of atleast a first VEGFR2-blocking, human anti-VEGF antibody of theinvention, or an antigen-binding fragment of the VEGFR2-blocking,anti-VEGF antibody of the invention, under conditions effective toinhibit VEGF-induced endothelial cell proliferation and/or migration.

Methods of, and uses in, inhibiting VEGFR2-induced macrophage functionare also provided, which generally comprise contacting a population ofcells or tissues that contains macrophages and VEGF with a compositioncomprising a biologically effective amount of at least a firstVEGFR2-blocking, human anti-VEGF antibody of the invention, or anantigen-binding fragment of the anti-VEGF antibody, under conditionseffective to inhibit VEGFR2-induced macrophage function.

The foregoing methods are preferably applied in the treatment of tumors,wherein the methods inhibit VEGFR2-induced macrophage function, therebyreducing the ability of tumor-infiltrating macrophages, which expressVEGFR2, to promote tumor progression and/or metastasis.

Methods of, and uses in, inhibiting VEGF-induced endothelial cellproliferation and/or migration and, optionally, angiogenesis, withoutsignificantly inhibiting VEGFR1-mediated stimulation of osteoclasts orchondroclasts are further provided. The methods generally comprisecontacting a population of cells or tissues that contain endothelialcells and at least one of osteoclasts or chondroclasts, with acomposition comprising a biologically effective amount of at least afirst VEGFR2-blocking, human anti-VEGF antibody of the invention, or anantigen-binding fragment of the antibody, under conditions effective toinhibit VEGF-induced endothelial cell proliferation and/or migration orangiogenesis, without significantly inhibiting VEGFR1-mediatedstimulation of osteoclasts or chondroclasts.

The foregoing methods and uses can be performed in vitro and in vivo, inthe latter case, wherein the tissues or cells are located within ananimal and the human anti-VEGF antibody is administered to the animal.In both cases, the methods and uses become methods and uses forinhibiting angiogenesis, comprising contacting a tissue comprising, or apopulation of, angiogenic or potentially angiogenic blood vessels, i.e.,those exposed to or potentially exposed to VEGF, with an anti-angiogeniccomposition comprising a biologically effective amount of at least afirst VEGFR2-blocking, anti-VEGF antibody of the invention, or anantigen-binding fragment thereof, under conditions effective to inhibitangiogenesis.

Where populations of potentially angiogenic blood vessels are maintainedex vivo, the present invention has utility in drug discovery programs.In vitro screening assays, with reliable positive and negative controls,are useful as a first step in the development of drugs to inhibit orpromoter angiogenesis, as well as in the delineation of furtherinformation on the angiogenic process. Where the population ofpotentially angiogenic blood vessels is located within an animal orpatient, the anti-angiogenic composition is administered to the animalas a form of therapy.

“Biologically effective amounts”, in terms of each of the foregoinginhibitory methods are therefore amounts of VEGFR2-blocking, humananti-VEGF antibodies of the invention, effective to inhibit VEGF-inducedendothelial cell proliferation and/or migration; to inhibit VEGF-inducedendothelial cell proliferation and/or migration, without significantlyinhibiting VEGFR1-induced cellular events; to inhibit VEGF-inducedendothelial cell proliferation and/or migration or angiogenesis, withoutsignificantly inhibiting VEGFR1 stimulation of osteoclasts orchondroclasts; and, overall, to reduce vascular endothelial cellproliferation and/or migration in a manner effective to inhibit bloodvessels growth or angiogenesis.

The invention thus provides methods of, and uses in, inhibitingVEGF-induced angiogenesis and, preferably, treating an angiogenicdisease, without significantly inhibiting VEGF stimulation ofosteoclasts or chondroclasts. The methods generally comprise contactinga population of cells or tissues that contain endothelial cells and atleast one of osteoclasts or chondroclasts, with a composition comprisinga biologically effective amount of at least a first VEGFR2-blocking,human anti-VEGF antibody of the invention, or an antigen-bindingfragment of the antibody, under conditions effective to inhibitVEGF-induced angiogenesis and to treat an angiogenic disease withoutsignificantly inhibiting VEGF stimulation of osteoclasts orchondroclasts.

Methods of, and uses in, inhibiting VEGF-induced angiogenesis and,preferably, treating an angiogenic disease, without causing significantside effects on bone metabolism are further provided. The methodsgenerally comprise contacting a tissue or a population of angiogenicvessels that contain vascular endothelial cells and at least one ofosteoclasts or chondroclasts, with a composition comprising abiologically effective amount of at least a first VEGFR2-blocking, humananti-VEGF antibody of the invention, or an antigen-binding fragment ofthe antibody, under conditions effective to inhibit VEGF-inducedangiogenesis and to treat an angiogenic disease without causingsignificant side effects on bone metabolism by not significantlyimpairing the activities of osteoclasts or chondroclasts.

Anti-angiogenic drug screening (in vitro) and therapy (in vivo) areprovided in terms of animals and patients that have, or are at risk fordeveloping, any disease or disorder characterized by undesired,inappropriate, aberrant, excessive and/or pathological vascularization.It is well known to those of ordinary skill in the art that as aberrantangiogenesis occurs in a wide range of diseases and disorders, a givenanti-angiogenic therapy, once shown to be effective in any acceptablemodel system, can be used to treat the entire range of diseases anddisorders connected with angiogenesis.

The methods and uses of the present invention are particularly intendedfor use in animals and patients that have, or are at risk fordeveloping, any form of vascularized tumor; macular degeneration,including age-related macular degeneration; arthritis, includingrheumatoid arthritis; atherosclerosis and atherosclerotic plaques;diabetic retinopathy and other retinopathies; thyroid hyperplasias,including Grave's disease; hemangioma; neovascular glaucoma; andpsoriasis.

The methods and uses of the invention are further intended for thetreatment of animals and patients that have, or are at risk fordeveloping, arteriovenous malformations (AVM), meningioma, and vascularrestenosis, including restenosis following angioplasty. Other intendedtargets of the therapeutic methods and uses are animals and patientsthat have, or are at risk for developing, angiofibroma, dermatitis,endometriosis, hemophilic joints, hypertrophic scars, inflammatorydiseases and disorders, pyogenic granuloma, scleroderma, synovitis,trachoma and vascular adhesions.

As disclosed in U.S. Pat. Nos. 5,712,291 and 6,524,583, eachspecifically incorporated herein by reference, each of the foregoingsomewhat preferred treatment groups are by no means exhaustive of thetypes of conditions that are to be treated by the present invention.U.S. Pat. Nos. 5,712,291 and 6,524,583 are each incorporated herein byreference for certain specific purposes, including the purpose ofidentifying a number of other conditions that may be effectively treatedby an anti-angiogenic therapeutic; the purpose of showing that thetreatment of all angiogenic diseases represents a unified concept, oncea defined category of angiogenesis-inhibiting compounds have beendisclosed and claimed (in the present case, VEGFR2-blocking, humananti-VEGF antibodies of the invention, and the purpose of showing thatthe treatment of all angiogenic diseases is enabled by data from only asingle model system.

In yet further aspects, and as disclosed in U.S. Pat. Nos. 5,712,291 and6,524,583, each incorporated herein by reference, the methods and usesof the present invention are intended for the treatment of animals andpatients that have, or are at risk for developing, abnormalproliferation of fibrovascular tissue, acne rosacea, acquired immunedeficiency syndrome, artery occlusion, atopic keratitis, bacterialulcers, Bechets disease, blood borne tumors, carotid obstructivedisease, chemical burns, choroidal neovascularization, chronicinflammation, chronic retinal detachment, chronic uveitis, chronicvitritis, contact lens overwear, corneal graft rejection, cornealneovascularization, corneal graft neovascularization, Crohn's disease,Eales disease, epidemic keratoconjunctivitis, fungal ulcers, Herpessimplex infections, Herpes zoster infections, hyperviscosity syndromes,Kaposi's sarcoma, leukemia, lipid degeneration, Lyme's disease, marginalkeratolysis, Mooren ulcer, Mycobacteria infections other than leprosy,myopia, ocular neovascular disease, optic pits, Osler-Weber syndrome(Osler-Weber-Rendu, osteoarthritis, Pagets disease, pars planitis,pemphigoid, phylectenulosis, polyarteritis, post-laser complications,protozoan infections, pseudoxanthoma elasticum, pterygium keratitissicca, radial keratotomy, retinal neovascularization, retinopathy ofprematurity, retrolental fibroplasias, sarcoid, scleritis, sickle cellanemia, Sogrens syndrome, solid tumors, Stargarts disease, Steven'sJohnson disease, superior limbic keratitis, syphilis, systemic lupus,Terrien's marginal degeneration, toxoplasmosis, trauma, tumors of Ewingsarcoma, tumors of neuroblastoma, tumors of osteosarcoma, tumors ofretinoblastoma, tumors of rhabdomyosarcoma, ulceritive colitis, veinocclusion, Vitamin A deficiency and Wegeners sarcoidosis.

The present invention further provides methods and uses for thetreatment of animals and patients that have, or are at risk fordeveloping, arthritis, in common with the treatment of arthritis usingimmunological agents described in U.S. Pat. No. 5,753,230, specificallyincorporated herein by reference. U.S. Pat. No. 5,972,922 is alsospecifically incorporated herein by reference to even further exemplifythe application of anti-angiogenic strategies to the treatment ofundesired angiogenesis associated with diabetes, parasitic diseases,abnormal wound healing, hypertrophy following surgery, burns, injury ortrauma, inhibition of hair growth, inhibition of ovulation and corpusluteum formation, inhibition of implantation and inhibition of embryodevelopment in the uterus. All of the foregoing conditions are thereforecontemplated for treatment by the methods and uses of the presentinvention.

U.S. Pat. No. 5,639,757 is further specifically incorporated herein byreference to exemplify the use of anti-angiogenic strategies to thegeneral treatment of graft rejection. The treatment of lunginflammation, nephrotic syndrome, preeclampsia, pericardial effusion,such as that associated with pericarditis, and pleural effusion usinganti-angiogenic strategies based upon VEGF inhibition is described in WO98/45331, specifically incorporated herein by reference. Animals andpatients that have, or are at risk for developing, any of the foregoingconditions are therefore contemplated for treatment by the methods anduses of the present invention.

As disclosed in WO 98/16551, specifically incorporated herein byreference, biological molecules that antagonize VEGF function are alsosuitable for use in treating diseases and disorders characterized byundesirable vascular permeability. Accordingly, the VEGF antagonizingantibodies, methods and uses of the present invention are applicable tothe treatment of animals and patients that have, or are at risk fordeveloping, diseases and disorders characterized by undesirable vascularpermeability, e.g., edema associated with brain tumors, ascitesassociated with malignancies, Meigs' syndrome, lung inflammation,nephrotic syndrome, pericardial effusion and pleural effusion and thelike.

Although the treatment of all the foregoing diseases is enabled withinthe present, unified invention, a particularly preferred aspect of themethods and uses of the present invention is application ofanti-angiogenic therapy to animals and patients that have, or are atrisk for developing, a vascularized solid tumor, a metastatic tumor ormetastases from a primary tumor.

Methods of, and uses in, inhibiting VEGF-induced angiogenesis, and,preferably, exerting an anti-tumor or improved anti-tumor effect withoutsignificantly inhibiting VEGF stimulation of osteoclasts orchondroclasts are further provided. The methods generally comprisecontacting a tissue, tumor environment or population of angiogenicvessels that contain vascular endothelial cells and at least one ofmacrophages, osteoclasts or chondroclasts, with a composition comprisinga biologically effective amount of at least a first VEGFR2-blocking,human anti-VEGF antibody of the invention, or an antigen-bindingfragment of the antibody, under conditions effective to inhibitVEGF-induced angiogenesis and to exert an anti-tumor or improvedanti-tumor effect without significantly inhibiting VEGF stimulation ofosteoclasts or chondroclasts.

The present invention thus further provides methods of, and uses in,treating a disease associated with angiogenesis, including all forms ofcancer associated with angiogenesis, comprising administering to ananimal or patient with such a disease or cancer a therapeuticallyeffective amount of at least a first pharmaceutical composition thatcomprises a VEGFR2-blocking, human anti-VEGF antibody of the invention,or an antigen-binding fragment or immunoconjugate of such an anti-VEGFantibody.

In addition, the methods and uses of the invention include methods anduses for inhibiting lymphangiogenesis, which comprise contacting atissue comprising, or a population of, lymphatic vessels (“lymphatics”),particularly, lymphatics exposed to or potentially exposed to VEGF, withan anti-angiogenic composition comprising a biologically effectiveamount of at least a first VEGFR2-blocking, anti-VEGF antibody of theinvention, or an antigen-binding fragment thereof, under conditionseffective to inhibit lymphangiogenesis.

Where populations of lymphatics are maintained ex vivo, the presentinvention has utility in drug discovery programs. Where the populationof lymphatics is located within an animal or patient, the composition ofthe invention is administered to the animal as a form of therapy.

In terms of inhibiting lymphangiogenesis, “biologically effectiveamounts” are amounts of VEGFR2-blocking, human anti-VEGF antibodies ofthe invention effective to inhibit VEGF-induced lymphangiogenesis, i.e.,VEGF-A stimulated lymphangiogenesis induced by VEGFR2. Preferably,VEGF-induced lymphangiogenesis will be induced without significantlyinhibiting VEGFR1-stimulated events, such as osteoclast or chondroclaststimulation.

The invention thus includes methods of, and uses in, treating a diseaseassociated with lymphangiogenesis, including all forms of cancerassociated with lymphangiogenesis, comprising administering to an animalor patient with such a disease or cancer a therapeutically effectiveamount of at least a first pharmaceutical composition that comprises aVEGFR2-blocking, human anti-VEGF antibody of the invention, or anantigen-binding fragment or immunoconjugate of such an anti-VEGFantibody.

A yet further aspect of the invention provides the use of the humanantibodies of the invention or an antigen-binding fragment orimmunoconjugate of such an antibody in the manufacture of a compositionor medicament for use in therapy, imaging or diagnosis.

A yet further aspect provides the human antibodies of the invention oran antigen-binding fragment or immunoconjugate of such an antibody foruse in therapy, diagnosis or imaging.

In addition, the invention provides compositions comprising the humanantibodies of the invention or an antigen-binding fragment orimmunoconjugate of such an antibody with one or more pharmaceuticallyacceptable excipient, carrier, diluent, buffer or stabilizer.

The in vivo methods as described herein are generally carried out in amammal. Any mammal may be treated, for example humans and any livestock,domestic or laboratory animal. Specific examples include mice, rats,pigs, cats, dogs, sheep, rabbits, cows and monkey. Preferably, however,the mammal is a human.

Thus, the term “animal” or “patient” as used herein includes any mammal,for example humans and any livestock, domestic or laboratory animal.Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits,cows and monkey. Preferably, however, the animal or patient is a humansubject.

This invention links both anti-angiogenic methods using unconjugated ornaked antibodies and fragments thereof, and vascular targeting methodsusing immunoconjugates in which a human antibody of the invention orantigen-binding fragment thereof, is operatively attached to atherapeutic agent. Unless otherwise specifically stated or made clear inscientific terms, the terms “antibody and fragment thereof”, as usedherein, therefore mean an “unconjugated or naked” human antibody orfragment, which is not attached to another agent, particularly atherapeutic or diagnostic agent. These definitions do not excludemodifications of the antibody, such as, by way of example only,modifications to improve the biological half life, affinity, avidity orother properties of the antibody, or combinations of the antibody withother effectors.

The anti-angiogenic treatment methods and uses of the invention alsoencompass the use of both unconjugated or naked antibodies andimmunoconjugates. In the immunoconjugate-based anti-angiogenic treatmentmethods, the human antibody of the invention, or antigen-bindingfragment thereof, is preferably operatively attached to a secondanti-angiogenic agent (the anti-VEGF antibody itself, being the firstanti-angiogenic agent). The attached anti-angiogenic agents may be thosethat have a direct or indirect anti-angiogenic effect.

The anti-angiogenic treatment methods and uses comprise administering toan animal or patient with a disease associated with angiogenesis,including all forms of cancer associated with angiogenesis, atherapeutically effective amount of at least a first pharmaceuticalcomposition that comprises at least a first unconjugated or nakedVEGFR2-blocking, human anti-VEGF antibody of the invention, orantigen-binding fragment thereof. Equally, the administered antibody maybe operatively associated with a second anti-angiogenic agent.

Methods for, and uses in, treating metastatic cancer compriseadministering to an animal or patient with metastatic cancer atherapeutically effective amount of at least a first pharmaceuticalcomposition that comprises at least a first unconjugated or nakedVEGFR2-blocking, human anti-VEGF antibody of the invention, orantigen-binding fragment thereof. Further methods are those wherein theadministered antibody may be operatively associated with a secondanti-angiogenic agent.

Methods for, and uses in, reducing metastases from a primary cancercomprise administering a therapeutically effective amount of at least afirst unconjugated or naked VEGFR2-blocking, human anti-VEGF antibody ofthe invention, or antigen-binding fragment thereof, to an animal orpatient that has, or was treated for, a primary cancer. Similarly, theadministered antibody may be operatively associated with a secondanti-angiogenic agent.

Methods for, and uses in, treating a disease associated withangiogenesis, including all forms of cancer associated withangiogenesis, further comprise administering to an animal or patientwith such a disease, e.g., a vascularized tumor, at least a firstunconjugated or naked VEGFR2-blocking, human anti-VEGF antibody of theinvention, or an antigen-binding fragment thereof, in an amounteffective to inhibit angiogenesis within the disease site orvascularized tumor. Equally, the administered antibody may beoperatively associated with a second anti-angiogenic agent.

The methods for, and uses in, treating a disease associated withangiogenesis, including all forms of cancer associated withangiogenesis, further comprise administering to an animal or patientwith such a disease or cancer at least a first unconjugated or nakedVEGFR2-blocking, human anti-VEGF antibody of the invention, or anantigen-binding fragment thereof, in an amount effective to inhibit VEGFbinding to the VEGF receptor VEGFR2 (KDR/Flk-1), thereby inhibitingangiogenesis within the disease or cancerous site. The administeredantibody may alternatively be operatively associated with a secondanti-angiogenic agent.

Methods for, and uses in, treating a disease associated withangiogenesis, including all forms of cancer associated withangiogenesis, also comprise administering to an animal or patient with avascularized tumor a therapeutically effective amount of at least afirst unconjugated or naked VEGFR2-blocking, human anti-VEGF antibody ofthe invention, or antigen-binding fragment thereof; wherein theanti-VEGF antibody substantially inhibits VEGF binding to the VEGFreceptor VEGFR2 (KDR/Flk-1) without significantly inhibiting VEGFbinding to the VEGF receptor VEGFR1 (Flt-1). Equally, the administeredantibody may be operatively associated with a second anti-angiogenicagent.

Yet further methods for, and uses in, treating a disease associated withangiogenesis, including all forms of cancer associated withangiogenesis, comprise administering to an animal or patient with such adisease, cancer or vascularized tumor a therapeutically effective amountof at least a first unconjugated or naked VEGFR2-blocking, humananti-VEGF antibody of the invention, or an antigen-binding fragmentthereof; wherein the anti-VEGF antibody substantially inhibits VEGFbinding to the VEGF receptor VEGFR2 (KDR/Flk-1) without significantlyinhibiting VEGF binding to the VEGF receptor VEGFR1 (Flt-1), therebyinhibiting angiogenesis within the disease site, cancer or vascularizedtumor without significantly impairing VEGFR1-mediated events in theanimal. The administered antibody may also be operatively associatedwith a second anti-angiogenic agent.

Additional methods for, and uses in, treating a disease associated withangiogenesis, including all forms of cancer associated withangiogenesis, comprise administering to an animal or patient with such adisease, cancer or vascularized tumor a therapeutically effective amountof at least a first unconjugated or naked VEGFR2-blocking, humananti-VEGF antibody of the invention, or an antigen-binding fragmentthereof; wherein the anti-VEGF antibody substantially inhibits VEGFbinding to the VEGF receptor VEGFR2 (KDR/Flk-1) without significantlyinhibiting VEGF binding to the VEGF receptor VEGFR1 (Flt-1), therebyinhibiting angiogenesis within the disease site, cancer or vascularizedtumor, including inhibiting VEGFR2-expressing macrophages in the diseasesite, particularly VEGFR2-expressing tumor-infiltrating macrophages. Theadministered antibody may also be operatively associated with a secondanti-angiogenic agent.

Still further methods for, and uses in, treating a disease associatedwith angiogenesis, including all forms of cancer associated withangiogenesis, comprise administering to an animal or patient with such adisease, cancer or vascularized tumor a therapeutically effective amountof at least a first unconjugated or naked VEGFR2-blocking, humananti-VEGF antibody of the invention, or an antigen-binding fragmentthereof; wherein the anti-VEGF antibody substantially inhibits VEGFbinding to the VEGF receptor VEGFR2 (KDR/Flk-1) without significantlyinhibiting VEGF binding to the VEGF receptor VEGFR1 (Flt-1), therebyinhibiting angiogenesis within the disease site, cancer or vascularizedtumor without significantly impairing osteoclast and/or chondroclastactivity in the animal. Equally, the administered antibody may beoperatively associated with a second anti-angiogenic agent.

Methods for, and uses in, treating a disease associated withangiogenesis, including all forms of cancer associated withangiogenesis, further comprise administering to an animal or patientwith such a disease, e.g., a vascularized tumor, at least a firstunconjugated or naked VEGFR2-blocking, human anti-VEGF antibody of theinvention, or an antigen-binding fragment thereof, in an amounteffective to inhibit angiogenesis within the disease site orvascularized tumor without exerting a significant adverse effect on bonemetabolism.

The foregoing anti-angiogenic treatment methods and uses will generallyinvolve the administration of the pharmaceutically effective compositionto the animal or patient systemically, such as by transdermal,intramuscular, intravenous injection and the like. However, any route ofadministration that allows the therapeutic agent to localize to theangiogenic site or sites, including tumor or intratumoral vascularendothelial cells, will be acceptable. Therefore, other suitable routesof delivery include oral, rectal, nasal, topical, and vaginal. U.S. Pat.No. 5,712,291, is specifically incorporated herein by reference forpurposes including further describing the various routes ofadministration that may be included in connection with the treatment ofan angiogenic disease or disorder.

For uses and methods for the treatment of arthritis, e.g., intrasynovialadministration may be employed, as described for other immunologicalagents in U.S. Pat. No. 5,753,230, specifically incorporated herein byreference. For conditions associated with the eye, ophthalmicformulations and administration are contemplated.

“Administration”, as used herein, means provision or delivery ofVEGFR2-blocking, human anti-VEGF antibody therapeutics in an amount(s)and for a period of time(s) effective to exert anti-angiogenic and/oranti-tumor effects. The passive administration of proteinaceoustherapeutics is generally preferred, in part, for its simplicity andreproducibility.

However, the term “administration” is herein used to refer to any andall means by which VEGFR2-blocking, anti-VEGF antibodies of theinvention are delivered or otherwise provided to the tumor vasculature.“Administration” therefore includes the provision of cells that producethe VEGFR2-blocking, human anti-VEGF antibody of the invention in amanner effective to result in delivery to the tumor. In suchembodiments, it may be desirable to formulate or package the cells in aselectively permeable membrane, structure or implantable device,generally one that can be removed to cease therapy. ExogenousVEGFR2-blocking, human anti-VEGF antibody of the invention will stillgenerally be preferred, as this represents a non-invasive method thatallows the dose to be closely monitored and controlled.

The therapeutic methods and uses of the invention also extend to theprovision of nucleic acids that encode a VEGFR2-blocking, humananti-VEGF antibody of the invention in a manner effective to result intheir expression in the vicinity of the tumor or their localization tothe tumor. Any gene therapy technique may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like.

In yet further embodiments, the invention provides methods for, and usesin, delivering selected therapeutic or diagnostic agents to angiogenicblood vessels associated with disease. Such embodiments are preferablyused for delivering selected therapeutic or diagnostic agents to tumoror intratumoral vasculature or stroma, and comprise administering to ananimal or patient having a vascularized tumor a biologically effectiveamount of a composition comprising at least a first immunoconjugate inwhich a diagnostic or therapeutic agent is operatively attached to aVEGFR2-blocking, human anti-VEGF antibody of the invention, orantigen-binding fragment thereof.

Although understanding the mechanism of action underlying the targetingaspects of the invention is not required in order to practice suchembodiments, it is believed that the antibodies of the invention deliverattached agents to angiogenic and tumor vasculature by virtue of bindingto VEGF bound to the VEGFR1 expressed thereon. These methods and uses ofthe invention thus concern delivering selected therapeutic or diagnosticagents to angiogenic blood vessels, tumor or intratumoral vasculature,and comprise administering to an animal or patient in need of treatmenta biologically effective amount of a composition comprising animmunoconjugate in which a diagnostic or therapeutic agent isoperatively attached to at least a first VEGFR2-blocking, humananti-VEGF antibody of the invention, or antigen-binding fragmentthereof, in a manner effective to allow binding of the antibody to VEGFbound to VEGFR1 expressed, overexpressed or upregulated on theangiogenic blood vessels, tumor or intratumoral vasculature, thusdelivering the diagnostic or therapeutic agent to the VEGF-VEGFR1 on theangiogenic blood vessels, tumor or intratumoral vasculature.

The delivery of selected therapeutic agents to tumor or intratumoralvasculature or stroma acts to arrest blood flow, or specifically arrestblood flow, in tumor vasculature; to destroy, or specifically destroy,tumor vasculature; and to induce necrosis, or specific necrosis in atumor. These methods and uses may thus be summarized as methods fortreating an animal or patient having a vascularized tumor, comprisingadministering to the animal or patient a therapeutically effectiveamount of at least a first pharmaceutical composition comprising atleast a first immunoconjugate that comprises a VEGFR2-blocking, humananti-VEGF antibody of the invention, or antigen-binding fragmentthereof, operatively attached to a therapeutic agent.

The “therapeutically effective amounts” for use in the invention areamounts of VEGFR2-blocking, human anti-VEGF antibody of the invention,or immunoconjugates thereof, effective to specifically kill at least aportion of tumor or intratumoral vascular endothelial cells; tospecifically induce apoptosis in at least a portion of tumor orintratumoral vascular endothelial cells; to specifically promotecoagulation in at least a portion of tumor or intratumoral bloodvessels; to specifically occlude or destroy at least a portion of bloodtransporting vessels of the tumor; to specifically induce necrosis in atleast a portion of a tumor; and/or to induce tumor regression orremission upon administration to selected animals or patients. Sucheffects are achieved while exhibiting little or no binding to, or littleor no killing of, vascular endothelial cells in normal, healthy tissues;little or no coagulation in, occlusion or destruction of blood vesselsin healthy, normal tissues; and exerting negligible or manageableadverse side effects on normal, healthy tissues of the animal orpatient.

The terms “preferentially” and “specifically”, as used herein in thecontext of promoting coagulation in, or destroying, tumor vasculature,and/or in the context of binding to tumor stroma and/or causing tumornecrosis, thus mean that the VEGFR2-blocking, human anti-VEGF antibodyof the invention or immunoconjugates thereof function to achieve stromalbinding, coagulation, destruction and/or tumor necrosis that issubstantially confined to the tumor stroma, vasculature and tumor site,and does not substantially extend to causing coagulation, destructionand/or tissue necrosis in normal, healthy tissues of the animal orsubject. The structure and function of healthy cells and tissues istherefore maintained substantially unimpaired by the practice of theinvention.

Although the antibodies of the invention effectively deliver agents toangiogenic and tumor vasculature by binding to VEGF in association withVEGFR1, other methods and uses operate on the basis of delivering atherapeutic agent to tumor stroma, wherein it exerts a therapeuticeffect on the nearby vessels. These methods and uses compriseadministering to an animal or patient with a vascularized tumor animmunoconjugate that comprises a therapeutic agent operatively attachedto at least a first VEGFR2-blocking, human anti-VEGF antibody of theinvention, or antigen-binding fragment thereof, in an amount effectiveto bind the immunoconjugate to non-receptor bound VEGF within the tumorstroma.

These methods and uses comprise administering to an animal or patientwith a vascularized tumor an immunoconjugate that comprises atherapeutic agent operatively attached to at least a firstVEGFR2-blocking, human anti-VEGF antibody of the invention, orantigen-binding fragment thereof, in an amount effective to localize theimmunoconjugate within the tumor stroma such that the attachedtherapeutic agent exerts an anti-tumor effect on the surrounding tumorvasculature and/or tumor cells.

The antibodies and compositions, as well as the methods and uses, of theinvention thus extend to compositions comprising VEGFR2-blocking,anti-VEGF antibodies comprising at least a first VEGFR2-blocking, humananti-VEGF antibody of the invention, or antigen-binding fragmentthereof, operatively attached to at least a first therapeutic ordiagnostic agent, more particularly, a first “distinct or exogenous”therapeutic agent. In this regard, the “VEGFR2-blocking, human anti-VEGFantibody” may itself be termed a “first therapeutic agent”. Accordingly,any attached therapeutic agent may be termed a first “distinct orexogenous therapeutic agent”, meaning that it is also a therapeuticagent, but distinct from and attached to the VEGFR2-blocking, humananti-VEGF antibody. Equivalent terminology for such conjugates is todescribe the at least a first VEGFR2-blocking, human anti-VEGF antibodyof the invention, or antigen-binding fragment thereof, as beingoperatively attached to at least a “second, distinct” therapeutic ordiagnostic agent.

VEGFR2-blocking, human anti-VEGF antibodies of the invention ortherapeutic conjugates are preferably linked to one or moreradiotherapeutic agents, anti-angiogenic agents, apoptosis-inducingagents, anti-tubulin drugs, anti-cellular or cytotoxic agents, orcoagulants (coagulation factors).

The invention thus provides a range of conjugated antibodies andfragments thereof in which the human antibody is operatively attached toat least a first therapeutic or diagnostic agent. The term“immunoconjugate” is broadly used to define the operative association ofthe antibody with another effective agent and is not intended to refersolely to any type of operative association, and is particularly notlimited to chemical “conjugation”. Recombinant fusion proteins areparticularly contemplated. So long as the delivery or targeting agent isable to bind to the target and the therapeutic or diagnostic agent issufficiently functional upon delivery, the mode of attachment will besuitable.

Attachment of agents via the carbohydrate moieties on antibodies is alsocontemplated. Glycosylation, both O-linked and N-linked, naturallyoccurs in antibodies. Recombinant antibodies can be modified to recreateor create additional glycosylation sites if desired, which is simplyachieved by engineering the appropriate amino acid sequences (such asAsn-X-Ser, Asn-X-Thr, Ser, or Thr) into the primary sequence of theantibody.

Currently preferred agents for use in VEGFR2-blocking, human anti-VEGFantibody or therapeutic conjugates of the invention and related methodsand uses are those that complement or enhance the effects of theantibody and/or those selected for a particular tumor type or patient.“Therapeutic agents that complement or enhance the effects of theantibody” include radiotherapeutic agents, anti-angiogenic agents,apoptosis-inducing agents and anti-tubulin drugs, any one or more ofwhich are preferred for use herewith.

The attachment or association of the preferred agents withVEGFR2-blocking, human anti-VEGF antibodies of the invention gives“immunoconjugates”, wherein such immunoconjugates often have enhancedand even synergistic anti-tumor properties. Currently preferredanti-angiogenic agents for use in this manner are angiostatin,endostatin, any one of the angiopoietins, vasculostatin, canstatin andmaspin. Currently preferred anti-tubulin drugs include colchicine,taxol, vinblastine, vincristine, vindescine and one or more of thecombretastatins.

The use of anti-cellular and cytotoxic agents results inVEGFR2-blocking, human anti-VEGF antibody “immunotoxins” of theinvention, whereas the use of coagulation factors results inVEGFR2-blocking, human anti-VEGF antibody or “coaguligands” of theinvention. The use of at least two therapeutic agents is alsocontemplated, such as combinations of one or more radiotherapeuticagents, anti-angiogenic agents, apoptosis-inducing agents, anti-tubulindrugs, anti-cellular and cytotoxic agents and coagulation factors.

In certain applications, the VEGFR2-blocking, human anti-VEGF antibodytherapeutics of the invention will be operatively attached to cytotoxic,cytostatic or otherwise anti-cellular agents that have the ability tokill or suppress the growth or cell division of endothelial cells.Suitable anti-cellular agents include chemotherapeutic agents, as wellas cytotoxins and cytostatic agents. Cytostatic agents are generallythose that disturb the natural cell cycle of a target cell, preferablyso that the cell is taken out of the cell cycle.

Exemplary chemotherapeutic agents include: steroids; cytokines;anti-metabolites, such as cytosine arabinoside, fluorouracil,methotrexate or aminopterin; anthracyclines; mitomycin C; vincaalkaloids; antibiotics; demecolcine; etoposide; mithramycin; andanti-tumor alkylating agents, such as chlorambucil or melphalan. Indeed,any of the agents disclosed herein in Table C could be used. Certainpreferred anti-cellular agents are DNA synthesis inhibitors, such asdaunorubicin, doxorubicin/adriamycin, and the like. Overall,taxol/paclitaxel, docetaxel, cisplatin, gemcitabine, a combretastatinand doxorubicin/adriamycin are currently preferred anti-cancer agents.

Of the cytokines and chemokines, currently preferred agents are IL-2,IL-12, TNF-α, interferon-α (IFN-α), IFN-β, IFN-γ, and LEC(liver-expressed chemokine). V-type ATPase inhibitors are also currentlypreferred, such as salicylihalamide, concanamycin or bafilomycin, as areprotein synthesis inhibitors, such as psymberin, pederin, irciniastatinA.

In certain therapeutic applications, toxin moieties will be preferred,due to the much greater ability of most toxins to deliver a cell killingeffect, as compared to other potential agents. Therefore, certainpreferred anti-cellular agents for VEGFR2-blocking, human anti-VEGFantibody constructs of the invention are plant-, fungus- orbacteria-derived toxins. Exemplary toxins include epipodophyllotoxins;bacterial endotoxin or the lipid A moiety of bacterial endotoxin;ribosome inactivating proteins, such as saporin or gelonin; a-sarcin;aspergillin; restrictocin; ribonucleases, such as placentalribonuclease; diphtheria toxin and pseudomonas exotoxin. Currentlypreferred examples are ricin, gelonin, abrin, diphtheria, pseudomonasand pertussis toxins.

Certain preferred toxins are the A chain toxins, such as ricin A chain.The most preferred toxin moiety is often ricin A chain that has beentreated to modify or remove carbohydrate residues, so called“deglycosylated A chain” (dgA). Deglycosylated ricin A chain ispreferred because of its extreme potency, longer half-life, and becauseit is economically feasible to manufacture it a clinical grade andscale. Recombinant and/or truncated ricin A chain may also be used.

For tumor targeting and treatment with immunotoxins, the followingpatents are specifically incorporated herein by reference for thepurposes of even further supplementing the present teachings regardinganti-cellular and cytotoxic agents: U.S. Pat. Nos. 6,004,554; 5,855,866;5,965,132; 5,776,427; 5,863,538; 5,660,827 and 6,051,230.

The VEGFR2-blocking, human anti-VEGF antibody of the present inventionmay be linked to an anti-tubulin drug. “Anti-tubulin drug(s)”, as usedherein, means any agent, drug, prodrug or combination thereof thatinhibits cell mitosis, preferably by directly or indirectly inhibitingtubulin activities necessary for cell mitosis, preferably tubulinpolymerization or depolymerization.

Currently preferred anti-tubulin drugs for use herewith are colchicine;taxanes, such as taxol, docetaxel and paclitaxel; vinca alkaloids, suchas vinblastine, vincristine and vindescine; and combretastatins.Exemplary combretastatins are combretastatin A, B and/or D, includingA-1, A-2, A-3, A-4, A-5, A-6, B-1, B-2, B-3, B-4, D-1 and D-2 andprodrug forms thereof.

The VEGFR2-blocking, human anti-VEGF antibody therapeutics of theinvention may comprise a component that is capable of promotingcoagulation, i.e., a coagulant. Here, the targeting antibody may bedirectly or indirectly, e.g., via another antibody, linked to a factorthat directly or indirectly stimulates coagulation.

Preferred coagulation factors for such uses are Tissue Factor (TF) andTF derivatives, such as truncated TF (tTF), dimeric, trimeric,polymeric/multimeric TF, and mutant TF deficient in the ability toactivate Factor VII. Other suitable coagulation factors include vitaminK-dependent coagulants, such as Factor II/IIa, Factor VII/VIIa, FactorIX/IXa and Factor X/Xa; vitamin K-dependent coagulation factors thatlack the Gla modification; Russell's viper venom Factor X activator;platelet-activating compounds, such as thromboxane A₂ and thromboxane A₂synthase; and inhibitors of fibrinolysis, such as α2-antiplasminOverall, truncated Tissue Factor (tTF) is currently preferred.

Tumor targeting and treatment with coaguligands is described in thefollowing patents, each of which are specifically incorporated herein byreference for the purposes of even further supplementing the presentteachings regarding coaguligands and coagulation factors: U.S. Pat. Nos.5,855,866; 5,965,132; 6,093,399; 6,004,555; 5,877,289; and 6,036,955.

The preparation of immunoconjugates and immunotoxins is generally wellknown in the art (see, e.g., U.S. Pat. No. 4,340,535). Each of thefollowing patents are further incorporated herein by reference for thepurposes of even further supplementing the present teachings regardingimmunotoxin generation, purification and use: U.S. Pat. Nos. 6,004,554;5,855,866; 5,965,132; 5,776,427; 5,863,538; 5,660,827 and 6,051,230.

In the preparation of immunoconjugates and immunotoxins, advantages maybe achieved through the use of certain linkers. For example, linkersthat contain a disulfide bond that is sterically “hindered” are oftenpreferred, due to their greater stability in vivo, thus preventingrelease of the toxin moiety prior to binding at the site of action. Itis generally desired to have a conjugate that will remain intact underconditions found everywhere in the body except the intended site ofaction, at which point it is desirable that the conjugate have good“release” characteristics.

Depending on the specific toxin compound used, it may be necessary toprovide a peptide spacer operatively attaching the VEGFR2-blocking,human anti-VEGF antibody of the invention and the toxin compound,wherein the peptide spacer is capable of folding into a disulfide-bondedloop structure. Proteolytic cleavage within the loop would then yield aheterodimeric polypeptide wherein the antibody and the toxin compoundare linked by only a single disulfide bond.

When certain other toxin compounds are utilized, a non-cleavable peptidespacer may be provided to operatively attach the VEGFR2-blocking, humananti-VEGF antibody of the invention and the toxin compound. Toxins thatmay be used in conjunction with non-cleavable peptide spacers are thosethat may, themselves, be converted by proteolytic cleavage, into acytotoxic disulfide-bonded form. An example of such a toxin compound isa Pseudomonas exotoxin compound.

A variety of chemotherapeutic and other pharmacological agents can alsobe successfully conjugated to VEGFR2-blocking, human anti-VEGF antibodytherapeutics of the invention. Exemplary antineoplastic agents that havebeen conjugated to antibodies include doxorubicin, daunomycin,methotrexate and vinblastine. Moreover, the attachment of other agentssuch as neocarzinostatin, macromycin, trenimon and α-amanitin has beendescribed (see U.S. Pat. Nos. 5,660,827; 5,855,866; and 5,965,132; eachincorporated herein.)

The preparation of coaguligands is also easily practiced. The operableassociation of one or more coagulation factors with a VEGFR2-blocking,human anti-VEGF antibody of the invention may be a direct linkage, suchas those described above for the immunotoxins. Alternatively, theoperative association may be an indirect attachment, such as where theantibody is operatively attached to a second binding region, preferablyan antibody or antigen binding region of an antibody, which binds to thecoagulation factor. The coagulation factor should be attached to theVEGFR2-blocking, human anti-VEGF antibody of the invention at a sitedistinct from its functional coagulating site, particularly where acovalent linkage is used to join the molecules.

Indirectly linked coaguligands are often based upon bispecificantibodies. The preparation of bispecific antibodies is also well knownin the art. One preparative method involves the separate preparation ofantibodies having specificity for the targeted tumor component, on theone hand, and the coagulating agent on the other. Peptic F(ab′γ)₂fragments from the two chosen antibodies are then generated, followed byreduction of each to provide separate Fab′γ_(SH) fragments. The SHgroups on one of the two partners to be coupled are then alkylated witha cross-linking reagent, such as o-phenylenedimaleimide, to provide freemaleimide groups on one partner. This partner may then be conjugated tothe other by means of a thioether linkage, to give the desired F(ab′γ)₂heteroconjugate (Glennie et al., 1987). Other approaches, such ascross-linking with SPDP or protein A may also be carried out.

In the preparation of immunoconjugates, immunotoxins and coaguligands,recombinant expression may be employed. The nucleic acid sequencesencoding the chosen VEGFR2-blocking, human anti-VEGF antibody of theinvention, and therapeutic agent, toxin or coagulant, are attachedin-frame in an expression vector. Recombinant expression thus results intranslation of the nucleic acid to yield the desired immunoconjugate.Chemical cross-linkers and avidin:biotin bridges may also join thetherapeutic agents to the VEGFR2-blocking, human anti-VEGF antibody ofthe invention.

The following patents are each incorporated herein by reference for thepurposes of even further supplementing the present teachings regardingcoaguligand preparation, purification and use, including bispecificantibody coaguligands: U.S. Pat. Nos. 5,855,866; 5,965,132; 6,093,399;6,004,555; 5,877,289; and 6,036,955.

Immunoconjugates with radiotherapeutic agents, anti-angiogenic agents,apoptosis-inducing agents, anti-tubulin drugs, toxins and coagulants,whether prepared by chemical conjugation or recombinant expression, mayemploy a biologically-releasable bond and/or a selectively cleavablespacer or linker Such compositions are preferably reasonably stableduring circulation and are preferentially or specifically released upondelivery to the disease or tumor site.

Certain preferred examples are acid sensitive spacers, whereinVEGFR2-blocking, human anti-VEGF antibodies of the invention linked tocolchicine or doxorubicin are particularly contemplated. Other preferredexamples are peptide linkers that include a cleavage site for peptidasesand/or proteinases that are specifically or preferentially present oractive within a disease site, such as a tumor environment. The deliveryof the immunoconjugate to the disease or tumor site results in cleavageand the relatively specific release of the coagulation factor.

Peptide linkers that include a cleavage site for urokinase,pro-urokinase, plasmin, plasminogen, TGFβ, staphylokinase, Thrombin,Factor IXa, Factor Xa or a metalloproteinase (MMP), such as aninterstitial collagenase, a gelatinase or a stromelysin, areparticularly preferred, as described and enabled by U.S. Pat. Nos.5,877,289 and 6,342,221, each incorporated herein by reference for suchpurposes.

The VEGFR2-blocking, human anti-VEGF antibody of the invention may alsobe derivatized to introduce functional groups permitting the attachmentof the therapeutic agent(s) through a biologically releasable bond. Thetargeting antibody may thus be derivatized to introduce side chainsterminating in hydrazide, hydrazine, primary amine or secondary aminegroups. Therapeutic agents may be conjugated through a Schiff's baselinkage, a hydrazone or acyl hydrazone bond or a hydrazide linker (U.S.Pat. Nos. 5,474,765 and 5,762,918).

Whether primarily anti-angiogenic or vascular-targeting based, thecompositions and methods of the present invention may be used incombination with other therapeutics and diagnostics. In terms ofbiological agents, preferably diagnostic or therapeutic agents, for use“in combination” with a VEGFR2-blocking, human anti-VEGF antibody inaccordance with the present invention, the term “in combination” issuccinctly used to cover a range of embodiments. The “in combination”terminology, unless otherwise specifically stated or made clear from thescientific terminology, thus applies to various formats of combinedcompositions, pharmaceuticals, cocktails, kits, methods, and first andsecond medical uses.

The “combined” embodiments of the invention thus include, for example,where the VEGFR2-blocking, human anti-VEGF of the invention is a nakedantibody and is used in combination with an agent or therapeutic agentthat is not operatively attached thereto. In such cases, the agent ortherapeutic agent may be used in a non-targeted or targeted form. In“non-targeted form”, the agent, particularly therapeutic agents, willgenerally be used according to their standard use in the art. In“targeted form”, the agent will generally be operatively attached to adistinct antibody or targeting region that delivers the agent ortherapeutic agent to the angiogenic disease site or tumor. The use ofsuch targeted forms of biological agents, both diagnostics andtherapeutics, is also quite standard in the art.

In other “combined” embodiments of the invention, the VEGFR2-blocking,human anti-VEGF antibody of the invention is an immunoconjugate whereinthe antibody is itself operatively associated or combined with the agentor therapeutic agent. The operative attachment includes all forms ofdirect and indirect attachment as described herein and known in the art.

The “combined” uses, particularly in terms of VEGFR2-blocking, humananti-VEGF antibody of the invention in combination with therapeuticagents, also include combined compositions, pharmaceuticals, cocktails,kits, methods, and first and second medical uses wherein the therapeuticagent is in the form of a prodrug. In such embodiments, the activatingcomponent able to convert the prodrug to the functional form of the drugmay again be operatively associated with the VEGFR2-blocking, humananti-VEGF antibodies of the present invention.

In certain preferred embodiments, the therapeutic compositions,combinations, pharmaceuticals, cocktails, kits, methods, and first andsecond medical uses will be “prodrug combinations”. As will beunderstood by those of ordinary skill in the art, the term “prodrugcombination”, unless otherwise stated, means that the antibody of theinvention is operatively attached to a component capable of convertingthe prodrug to the active drug, not that the antibody is attached to theprodrug itself. However, there is no requirement that the prodrugembodiments of the invention need to be used as prodrug combinations.Accordingly, prodrugs may be used in any manner that they are used inthe art, including in ADEPT and other forms.

Thus, where combined compositions, pharmaceuticals, cocktails, kits,methods, and first and second medical uses are described, preferably interms of diagnostic agents, and more preferably therapeutic agents, thecombinations include VEGFR2-blocking, human anti-VEGF antibodies thatare naked antibodies and immunoconjugates, and wherein practice of thein vivo embodiments of the invention involves the prior, simultaneous orsubsequent administration of the naked antibodies or immunoconjugate andthe biological, diagnostic or therapeutic agent; so long as, in someconjugated or unconjugated form, the overall provision of some form ofthe antibody and some form of the biological, diagnostic or therapeuticagent is achieved.

Particularly preferred combined compositions, methods and uses of theinvention are those including VEGFR2-blocking, human anti-VEGFantibodies of the invention and endostatin (U.S. Pat. No. 5,854,205).These include where the VEGFR2-blocking, human anti-VEGF antibody of theinvention is a naked antibody or immunoconjugate; and when animmunoconjugate, wherein the VEGFR2-blocking, human anti-VEGF antibodyof the invention is linked to endostatin, optionally with angiostatin;wherein the combined therapeutic method or use involves the prior,simultaneous, or subsequent administration of endostatin, optionallywith angiostatin; so long as, in some conjugated or unconjugated form,the overall provision of the antibody, endostatin and optionallyangiostatin is achieved. VEGFR2-blocking, human anti-VEGF antibodies ofthe invention operatively associated with collagenase are also provided,as the collagenase, when specifically delivered to the tumor, willproduce endostatin in situ, achieving similar benefits.

The foregoing and other explanations of the effects of the presentinvention on tumors are made for simplicity to explain the combined modeof operation, type of attached agent(s) and such like. This descriptiveapproach should not be interpreted as either an understatement or anoversimplification of the beneficial properties of the VEGFR2-blocking,human anti-VEGF antibodies of the invention. It will therefore beunderstood that such antibodies themselves have anti-angiogenicproperties and VEGF neutralization properties (such as neutralizing thesurvival function of VEGF), that immunoconjugates of such antibodieswill maintain these properties and combine them with the properties ofthe attached agent; and further, that the combined effect of theantibody and any attached agent will typically be enhanced and/ormagnified.

The invention therefore provides compositions, pharmaceuticalcompositions, therapeutic kits and medicinal cocktails comprising,optionally in at least a first composition or container, a biologicallyeffective amount of at least a first VEGFR2-blocking, human anti-VEGFantibody of the invention, or an antigen-binding fragment orimmunoconjugate of such an anti-VEGF antibody; and a biologicallyeffective amount of at least a second biological agent, component orsystem.

The “at least a second biological agent, component or system” will oftenbe a therapeutic or diagnostic agent, component or system, but it notbe. For example, the at least a second biological agent, component orsystem may comprise components for modification of the antibody and/orfor attaching other agents to the antibody. Certain preferred secondbiological agents, components or systems are prodrugs or components formaking and using prodrugs, including components for making the prodrugitself and components for adapting the antibodies of the invention tofunction in such prodrug or ADEPT embodiments.

Where therapeutic or diagnostic agents are included as the at least asecond biological agent, component or system, such therapeutics and/ordiagnostics will typically be those for use in connection withangiogenic diseases. Such agents are those suitable for use in treatingor diagnosing a disease or disorder as disclosed in any one of U.S. Pat.Nos. 5,712,291, 5,753,230, 5,972,922, 5,639,757, WO 98/45331 and WO98/16551, each specifically incorporated herein by reference.

Where the disease to be treated is cancer, “at least a secondanti-cancer agent” will be included in the therapeutic kit or cocktail.The term “at least a second anti-cancer agent” is chosen in reference tothe VEGFR2-blocking, human anti-VEGF antibody of the invention being thefirst anti-cancer agent. The antibodies of the invention may thus becombined with chemotherapeutic agents, radiotherapeutic agents,cytokines, anti-angiogenic agents, apoptosis-inducing agents oranti-cancer immunotoxins or coaguligands.

“Chemotherapeutic agents”, as used herein, refer to classicalchemotherapeutic agents or drugs used in the treatment of malignancies.This term is used for simplicity notwithstanding the fact that othercompounds may be technically described as chemotherapeutic agents inthat they exert an anti-cancer effect. However, “chemotherapeutic” hascome to have a distinct meaning in the art and is being used accordingto this standard meaning. A number of exemplary chemotherapeutic agentsare described herein. Those of ordinary skill in the art will readilyunderstand the uses and appropriate doses of chemotherapeutic agents,although the doses may well be reduced when used in combination with thepresent invention.

A new class of drugs that may also be termed “chemotherapeutic agents”are agents that induce apoptosis. Any one or more of such drugs,including genes, vectors, antisense constructs and ribozymes, asappropriate, may also be used in conjunction with the present invention.Currently preferred second agents are anti-angiogenic agents, such asangiostatin, endostatin, vasculostatin, canstatin and maspin.

Other exemplary anti-cancer agent include, e.g., neomycin,podophyllotoxin(s), TNF-α, α_(v)β₃ antagonists, calcium ionophores,calcium-flux inducing agents, and any derivative or prodrug thereof.Currently preferred anti-tubulin drugs include colchicine, taxol,vinblastine, vincristine, vindescine, a combretastatin or a derivativeor prodrug thereof.

Anti-cancer immunotoxins or coaguligands are further appropriateanti-cancer agents. “Anti-cancer immunotoxins or coaguligands”, ortargeting-agent/therapeutic agent constructs, are based upon targetingagents, including antibodies or antigen binding fragments thereof, thatbind to a targetable or accessible component of a tumor cell, tumorvasculature or tumor stroma, and that are operatively attached to atherapeutic agent, including cytotoxic agents (immunotoxins) andcoagulation factors (coaguligands). A “targetable or accessiblecomponent” of a tumor cell, tumor vasculature or tumor stroma, ispreferably a surface-expressed, surface-accessible or surface-localizedcomponent, although components released from necrotic or otherwisedamaged tumor cells or vascular endothelial cells may also be targeted,including cytosolic and/or nuclear tumor cell antigens.

Both antibody and non-antibody targeting agents may be used, includinggrowth factors, such as VEGF and FGF; peptides containing the tripeptideR-G-D, that bind specifically to the tumor vasculature; and othertargeting components such as annexins and related ligands.

Anti-tumor cell immunotoxins or coaguligands may comprise antibodiesexemplified by the group consisting of antibodies termed B3 (ATCC HB10573), 260F9 (ATCC HB 8488), D612 (ATCC HB 9796) and KS1/4, said KS1/4antibody obtained from a cell comprising the vector pGKC2310 (NRRLB-18356) or the vector pG2A52 (NRRL B-18357).

Anti-tumor cell targeting agents that comprise an antibody, or anantigen-binding region thereof, that binds to an intracellular componentthat is released from a necrotic tumor cell are also contemplated.Preferably such antibodies are monoclonal antibodies, or antigen-bindingfragments thereof, that bind to insoluble intracellular antigen(s)present in cells that may be induced to be permeable, or in cell ghostsof substantially all neoplastic and normal cells, but are not present oraccessible on the exterior of normal living cells of a mammal

U.S. Pat. Nos. 5,019,368, 4,861,581 and 5,882,626, each issued to AlanEpstein and colleagues, are each specifically incorporated herein byreference for purposes of even further describing and teaching how tomake and use antibodies specific for intracellular antigens that becomeaccessible from malignant cells in vivo. The antibodies described aresufficiently specific to internal cellular components of mammalianmalignant cells, but not to external cellular components. Exemplarytargets include histones, but all intracellular components specificallyreleased from necrotic tumor cells are encompassed.

Upon administration to an animal or patient with a vascularized tumor,such antibodies localize to the malignant cells by virtue of the factthat vascularized tumors naturally contain necrotic tumor cells, due tothe process(es) of tumor re-modeling that occur in vivo and cause atleast a proportion of malignant cells to become necrotic. In addition,the use of such antibodies in combination with other therapies thatenhance tumor necrosis serves to enhance the effectiveness of targetingand subsequent therapy.

These types of antibodies may thus be used to directly or indirectlyassociate with angiopoietins and to administer the angiopoietins tonecrotic malignant cells within vascularized tumors, as genericallydisclosed herein.

As also disclosed in U.S. Pat. Nos. 5,019,368, 4,861,581 and 5,882,626,each incorporated herein by reference, these antibodies may be used incombined diagnostic methods (see below) and in methods for measuring theeffectiveness of anti-tumor therapies. Such methods generally involvethe preparation and administration of a labeled version of theantibodies and measuring the binding of the labeled antibody to theinternal cellular component target preferentially bound within necrotictissue. The methods thereby image the necrotic tissue, wherein alocalized concentration of the antibody is indicative of the presence ofa tumor and indicate ghosts of cells that have been killed by theanti-tumor therapy.

Anti-tumor stroma immunotoxins or coaguligands will generally compriseantibodies that bind to a connective tissue component, a basementmembrane component or an activated platelet component; as exemplified bybinding to fibrin, RIBS or FIBS.

Anti-tumor vasculature immunotoxins or coaguligands may compriseligands, antibodies, or fragments thereof, which bind to asurface-expressed, surface-accessible or surface-localized component ofthe blood transporting vessels, preferably the intratumoral bloodvessels, of a vascularized tumor. Such antibodies include those thatbind to surface-expressed components of intratumoral blood vessels of avascularized tumor, including intratumoral vasculature cell surfacereceptors, such as endoglin (TEC-4 and TEC-11 antibodies), a TGFβreceptor, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a VEGF/VPFreceptor, an FGF receptor, a TIE, α_(v)β₃ integrin, pleiotropin,endosialin and MHC Class II proteins. The antibodies may also bind tocytokine-inducible or coagulant-inducible components of intratumoralblood vessels. Certain preferred agents will bind to aminophospholipids,such as phosphatidylserine or phosphatidylethanolamine.

Other anti-tumor vasculature immunotoxins or coaguligands may compriseantibodies, or fragments thereof, that bind to a ligand or growth factorthat binds to an intratumoral vasculature cell surface receptor. Suchantibodies include those that bind to VEGF/VPF (GV39 and GV97antibodies), FGF, TGFβ, a ligand that binds to a TIE, a tumor-associatedfibronectin isoform, scatter factor/hepatocyte growth factor (HGF),platelet factor 4 (PF4), PDGF and TIMP. The antibodies, or fragmentsthereof, may also bind to a ligand:receptor complex or a growthfactor:receptor complex, but not to the ligand or growth factor, or tothe receptor, when the ligand or growth factor or the receptor is not inthe ligand:receptor or growth factor:receptor complex.

Anti-tumor cell, anti-tumor stroma or anti-tumor vasculatureantibody-therapeutic agent constructs may comprise anti-angiogenicagents, apoptosis-inducing agents, anti-tubulin drugs, cytotoxic agentssuch as plant-, fungus- or bacteria-derived toxins. Ricin A chain anddeglycosylated ricin A chain will often be preferred. Anti-tumor cell,anti-tumor stroma or anti-tumor vasculature antibody-therapeutic agentconstructs may comprise coagulants (direct and indirect actingcoagulation factors) or second antibody binding regions that bind tocoagulation factors. The operative association with Tissue Factor orTissue Factor derivatives, such as truncated Tissue Factor, will oftenbe preferred.

In terms of compositions, kits and/or medicaments of the invention, thecombined effective amounts of the therapeutic agents may be comprisedwithin a single container or container means, or comprised withindistinct containers or container means. The cocktails will generally beadmixed together for combined use. Agents formulated for intravenousadministration will often be preferred. Imaging components may also beincluded. The kits may also comprise instructions for using the at leasta first antibody and the one or more other biological agents included.

Speaking generally, the at least a second anti-cancer agent may beadministered to the animal or patient substantially simultaneously withthe VEGFR2-blocking, human anti-VEGF antibody of the invention; such asfrom a single pharmaceutical composition or from two pharmaceuticalcompositions administered closely together.

Alternatively, the at least a second anti-cancer agent may beadministered to the animal or patient at a time sequential to theadministration of the VEGFR2-blocking, human anti-VEGF antibody of theinvention. “At a time sequential”, as used herein, means “staggered”,such that the at least a second anti-cancer agent is administered to theanimal or patient at a time distinct to the administration of theVEGFR2-blocking, human anti-VEGF antibody of the invention. Generally,the two agents are administered at times effectively spaced apart toallow the two agents to exert their respective therapeutic effects,i.e., they are administered at “biologically effective time intervals”.The at least a second anti-cancer agent may be administered to theanimal or patient at a biologically effective time prior to theVEGFR2-blocking, human anti-VEGF antibody of the invention, or at abiologically effective time subsequent to that therapeutic.

Accordingly, the present invention provides methods for treating ananimal or patient with a vascularized tumor, comprising:

-   -   (a) subjecting the animal or patient to a first treatment that        substantially reduces the tumor burden; and    -   (b) subsequently administering at least a first anti-angiogenic        agent to the animal or patient in an amount effective to inhibit        metastasis from any surviving tumor cells; wherein the first        anti-angiogenic agent is at least a first VEGFR2-blocking, human        anti-VEGF antibody of the invention, or antigen-binding fragment        thereof; optionally wherein the antibody or fragment is        operatively associated with a second anti-angiogenic agent.

Preferred first treatments include surgical resection andchemotherapeutic intervention. Combined anti-angiogenics can also beused. Other treatment methods for animals or patients with vascularizedtumors, comprise:

-   -   (a) administering a first antibody-therapeutic agent construct        to the animal or patient in an amount effective to induce        substantial tumor necrosis; wherein the first        antibody-therapeutic agent construct comprises a therapeutic        agent operatively linked to a first antibody, or antigen binding        fragment thereof, that binds to a surface-expressed,        surface-accessible or surface-localized component of a tumor        cell, tumor vasculature or tumor stroma; and    -   (b) subsequently administering a second antibody to the animal        or patient in an amount effective to inhibit metastasis from any        surviving tumor cells; wherein the second antibody is at least a        first VEGFR2-blocking, human anti-VEGF antibody of the        invention, or antigen-binding fragment thereof; and further        optionally wherein the antibody or fragment is operatively        associated with a second anti-angiogenic agent.

In particularly preferred embodiments, human VEGFR2-blocking, anti-VEGFantibodies of the invention are provided for use in combination withprodrugs and ADEPT. In such compositions, combination, pharmaceuticals,kits, methods and uses, the VEGFR2-blocking, human anti-VEGF antibody ofthe invention or fragment thereof will be modified to provide aconverting or enzymatic capacity, or operatively associated with,preferably covalently linked or conjugated to, at least a firstconverting agent or enzyme capable of converting at least one prodrug tothe active form of the drug.

The enzymatic or enzyme-conjugated antibody or fragment will combinedwith an initially separate formulation of the “prodrug”. The prodrugwill be an inactive or weakly active form of a drug that is that isconverted to the active form of the drug on contact with the enzymaticcapacity, converting function or enzyme associated with theVEGFR2-blocking, human anti-VEGF of the invention.

Accordingly, kits are provided that comprise, preferably in separatecompositions and/or containers:

-   -   (a) a biologically effective amount of at least a first        VEGFR2-blocking, human anti-VEGF antibody of the invention or        fragment thereof, that has an enzymatic function, preferably        where the antibody or fragment is operatively associated with,        covalently linked or conjugated to, at least a first enzyme; and    -   (b) a biologically effective amount of at least a first        substantially inactive prodrug that is converted to a        substantially active drug by the enzymatic function of, or        enzyme associated with, linked to or conjugated to the        VEGFR2-blocking, human anti-VEGF antibody or fragment.

The present invention further provides advantageous methods and usesthat comprise:

-   -   (a) administering to an animal or patient with a vascularized        tumor a biologically effective amount of at least a first        pharmaceutical composition comprising at least a first        VEGFR2-blocking, human anti-VEGF antibody of the invention, or        antigen binding fragment thereof, wherein the antibody or        fragment has an enzymatic function, preferably wherein the        antibody or fragment is operatively associated with, covalently        linked to, or conjugated to, at least a first enzyme; wherein        said antibody or fragment localizes to the vasculature,        intratumoral vasculature or stroma of the vascularized tumor        after administration; and    -   (b) subsequently administering to the animal or patient, after        an effective time period, a biologically effective amount of at        least a second pharmaceutical composition comprising a        biologically effective amount of at least one substantially        inactive prodrug; wherein the prodrug is converted to a        substantially active drug by the enzymatic function of, or        enzyme associated with, linked to, or conjugated to the        VEGFR2-blocking, human anti-VEGF antibody or fragment of the        invention localized within the vasculature, intratumoral        vasculature or stroma of said vascularized tumor.

In certain other embodiments, the antibodies and immunoconjugates of theinvention may be combined with one or more diagnostic agents, typicallydiagnostic agents for use in connection with angiogenic diseases. Arange of diagnostic compositions, kits and methods are thus includedwithin the invention.

Yet further aspects are methods of diagnosis or imaging of a subjectcomprising the administration of an appropriate amount of a humanantibody or other protein of the invention as defined herein to thesubject and detecting the presence and/or amount and/or the location ofthe antibody or other protein of the invention in the subject.

Appropriate diseases to be imaged or diagnosed in accordance with theabove described uses and methods include any disease associated withangiogenesis as described elsewhere herein.

In one embodiment, the invention provides a method of diagnosing adisease associated with angiogenesis in a mammal comprising the step of:

-   -   (a) contacting a test sample taken from said mammal with any one        or more of the antibodies of the invention.

In a further embodiment, the invention provides a method of diagnosingdisease associated with angiogenesis in a mammal comprising the stepsof:

-   -   (a) contacting a test sample taken from said mammal with one or        more of the antibodies of the invention;    -   (b) measuring the presence and/or amount and/or location of        antibody-antigen complex in the test sample; and, optionally    -   (c) comparing the presence and/or amount of antibody-antigen        complex in the test sample to a control.

In the above methods, said contacting step is carried out underconditions that permit the formation of an antibody-antigen complex.Appropriate conditions can readily be determined by a person skilled inthe art.

In the above methods any appropriate test sample may be used, forexample biopsy cells, tissues or organs suspected of being affected bydisease or histological sections.

In certain of the above methods, the presence of any amount ofantibody-antigen complex in the test sample would be indicative of thepresence of disease. Preferably, for a positive diagnosis to be made,the amount of antibody-antigen complex in the test sample is greaterthan, preferably significantly greater than, the amount found in anappropriate control sample. More preferably, the significantly greaterlevels are statistically significant, preferably with a probabilityvalue of <0.05. Appropriate methods of determining statisticalsignificance are well known and documented in the art and any of thesemay be used.

Appropriate control samples could be readily chosen by a person skilledin the art, for example, in the case of diagnosis of a particulardisease, an appropriate control would be a sample from a subject thatdid not have that disease. Appropriate control “values” could also bereadily determined without running a control “sample” in every test,e.g., by reference to the range for normal subjects known in the art.

For use in the diagnostic or imaging applications, the antibodies of theinvention may be labeled with a detectable marker such as a radio-opaqueor radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²³I, ¹²⁵I, ¹³¹I; aradioactive emitter (e.g., α, β or γ emitters); a fluorescent(fluorophore) or chemiluminescent (chromophore) compound, such asfluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such asalkaline phosphatase, beta-galactosidase or horseradish peroxidase; animaging agent; or a metal ion; or a chemical moiety such as biotin whichmay be detected by binding to a specific cognate detectable moiety,e.g., labelled avidin/streptavidin. Methods of attaching a label to abinding protein, such as an antibody or antibody fragment, are known inthe art. Such detectable markers allow the presence, amount or locationof binding protein-antigen complexes in the test sample to be examined.

Preferred detectable markers for in vivo use include an X-ray detectablecompound, such as bismuth (III), gold (III), lanthanum (III) or lead(II); a radioactive ion, such as copper⁶⁷, gallium⁶⁷, gallium⁶⁸,indium¹¹¹, indium¹¹³, iodine¹²³, iodine¹²⁵, iodine¹³¹, mercury¹⁹⁷,mercury²⁰³, rhenium¹⁸⁶, rhenium¹⁸⁸, rubidium⁹⁷, rubidium¹⁰³,technetium^(99m) or yttrium⁹⁰; a nuclear magnetic spin-resonanceisotope, such as cobalt (II), copper (II), chromium (III), dysprosium(III), erbium (III), gadolinium (III), holmium (III), iron (II), iron(III), manganese (II), neodymium (III), nickel (II), samarium (III),terbium (III), vanadium (II) or ytterbium (III); or rhodamine orfluorescein.

The invention also includes diagnostic or imaging agents comprising theantibodies of the invention attached to a label that produces adetectable signal, directly or indirectly. Appropriate labels aredescribed elsewhere herein.

The invention further includes kits comprising one or more of the humanantibodies or compositions of the invention or one or more of thenucleic acid molecules encoding the antibodies of the invention, or oneor more recombinant expression vectors comprising the nucleic acidsequences of the invention, or one or more host cells or virusescomprising the recombinant expression vectors or nucleic acid sequencesof the invention. Preferably said kits are for use in the methods anduses as described herein, e.g., the therapeutic, diagnostic or imagingmethods as described herein, or are for use in the in vitro assays ormethods as described herein. The antibody in such kits may preferably bean antibody conjugate as described elsewhere herein, e.g., may beconjugated to a detectable moiety or may be an immumoconjugate.Preferably said kits comprise instructions for use of the kitcomponents, for example in diagnosis. Preferably said kits are fordiagnosing diseases associated with angiogenesis and optionally compriseinstructions for use of the kit components to diagnose such diseases.

The antibodies of the invention as defined herein may also be used asmolecular tools for in vitro or in vivo applications and assays. As theantibodies have an antigen binding site, these can function as membersof specific binding pairs and these molecules can be used in any assaywhere the particular binding pair member is required.

Thus, yet further aspects of the invention provide a reagent thatcomprises an antibody of the invention as defined herein and the use ofsuch antibodies as molecular tools, for example in in vitro or in vivoassays.

In terms of cancer diagnosis and treatment, the diagnostic and imagingcompositions, kits and methods of the present invention include in vivoand in vitro diagnostics. For example, a vascularized tumor may beimaged using a diagnostically effective amount of a tumor diagnosticcomponent that comprises at least a first binding region that binds toan accessible component of a tumor cell, tumor vasculature or tumorstroma, operatively attached to an in vivo diagnostic imaging agent.

The tumor imaging is preferably conducted to provide an image of thestroma and/or vasculature of a vascularized tumor using a diagnosticcomponent that comprises at least a first binding region that binds toan accessible component of tumor vasculature or tumor stroma. Anysuitable binding region or antibody may be employed, such as thosedescribed above in terms of the therapeutic constructs. Certainadvantages will be provided by using a detectably-labeledVEGFR2-blocking, human anti-VEGF antibody of the invention construct,wherein the image formed will be predictive of the binding sites of thetherapeutic to be used.

Detectably-labeled in vivo tumor diagnostics, preferably aVEGFR2-blocking, human anti-VEGF antibody of the invention, may comprisean X-ray detectable compound, such as bismuth (III), gold (III),lanthanum (III) or lead (II); a radioactive ion, such as copper⁶⁷,gallium⁶⁷, gallium⁶⁸, indium¹¹¹, indium¹¹³, iodine¹²³, iodine¹²⁵,iodine¹³¹, mercury¹⁹⁷, mercury²⁰³, rhenium¹⁸⁶, rhenium¹⁸⁸, rubidium⁹⁷,rubidium¹⁰³, technetium^(99m) or yttrium⁹⁰; a nuclear magneticspin-resonance isotope, such as cobalt (II), copper (II), chromium(III), dysprosium (III), erbium (III), gadolinium (III), holmium (III),iron (II), iron (III), manganese (II), neodymium (III), nickel (II),samarium (III), terbium (III), vanadium (II) or ytterbium (III); orrhodamine or fluorescein.

Pre-imaging before tumor treatment may be carried out by:

-   -   (a) administering to the animal or patient a diagnostically        effective amount of a pharmaceutical composition comprising a        diagnostic agent operatively attached to at least a first        binding region that binds to an accessible component of a tumor        cell, tumor vasculature (preferably) or tumor stroma        (preferably), including diagnostic agents operatively attached        to a VEGFR2-blocking, human anti-VEGF antibody construct of the        invention; and    -   (b) subsequently detecting the detectably-labeled first binding        region (or VEGFR2-blocking, human anti-VEGF antibody of the        invention) bound to the tumor cells, tumor blood vessels        (preferably) or tumor stroma (preferably); thereby obtaining an        image of the tumor, tumor vasculature and/or tumor stroma.

Cancer treatment may also be carried out by:

-   -   (a) forming an image of a vascularized tumor by administering to        an animal or patient having a vascularized tumor a        diagnostically minimal amount of at least a first        detectably-labeled tumor binding agent, preferably a        VEGFR2-blocking, human anti-VEGF antibody construct of the        invention, comprising a diagnostic agent operatively attached to        the tumor binding agent or VEGFR2-blocking, anti-VEGF antibody        of the invention, thereby forming a detectable image of the        tumor, tumor vasculature (preferably), or tumor stroma        (preferably); and    -   (b) subsequently administering to the same animal or patient a        therapeutically optimized amount of at least a first naked        VEGFR2-blocking, human anti-VEGF antibody of the invention or        therapeutic agent-antibody construct using such an antibody,        thereby causing an anti-tumor effect.

Imaging and treatment formulations or medicaments are thus provided,which generally comprise:

-   -   (a) a first pharmaceutical composition comprising a        diagnostically effective amount of a detectably-labeled tumor        binding agent, preferably a VEGFR2-blocking, human anti-VEGF        antibody construct of the invention, that comprises a detectable        agent operatively attached to the tumor binding agent or        VEGFR2-blocking, human anti-VEGF antibody of the invention; and    -   (b) a second pharmaceutical composition comprising a        therapeutically effective amount of at least one naked        VEGFR2-blocking, human anti-VEGF antibody of the invention or        therapeutic agent-antibody construct using such an antibody.

The invention also provides in vitro diagnostic kits comprising at leasta first composition or pharmaceutical composition comprising abiologically effective amount of at least one diagnostic agent that isoperatively associated with at least a first VEGFR2-blocking, humananti-VEGF antibody of the invention, or an antigen-binding fragmentthereof.

The invention still further provides combined kits in which thediagnostic agent is intended for use outside the body, preferably inconnection with a test conducted on a biological sample obtained from ananimal or patient. As such, the invention provides kits comprising,generally in at least two distinct containers, at least a firstcomposition, pharmaceutical composition or medicinal cocktail comprisinga biologically effective amount of at least a first VEGFR2-blocking,human anti-VEGF antibody of the invention, or an antigen-bindingfragment or immunoconjugate of such an anti-VEGF antibody; and abiologically effective amount of at least one diagnostic agent,component or system for in vitro use.

The “diagnostic agent, component or system for in vitro use” will be anydiagnostic agent or combination of agents that allow the diagnosis ofone or more diseases that have an angiogenic component. The in vitrodiagnostics thus include those suitable for use in generating diagnosticor prognostic information in relation to a disease or disorder asdisclosed in any one of U.S. Pat. Nos. 5,712,291, 5,753,230, 5,972,922,5,639,757, WO 98/45331 and WO 98/16551, each specifically incorporatedherein by reference.

In terms of cancer diagnosis and treatment, the in vitro diagnosticswill preferably include a diagnostic component that comprises at least afirst binding region that binds to an accessible component of a tumorcell, tumor vasculature (preferably) or tumor stroma (preferably)operatively attached to a “detectable or reporter agent” directly orindirectly detectable by an in vitro diagnostic test. “Detectable orreporter agents” directly detectable in vitro include those such asradiolabels and reporter agents detectable by immunofluorescence.

“Detectable or reporter agents” indirectly detectable in vitro includethose that function in conjunction with further exogenous agent(s), suchas detectable enzymes that yield a colored product on contact with achromogenic substrate. Indirect detection in vitro also extends todetectable or reporter components or systems that comprise the firstbinding region that binds to an accessible component of a tumor cell,tumor vasculature (preferably) or tumor stroma (preferably) incombination with at least one detecting antibody that hasimmunospecificity for the first binding region. The “detecting antibody”is preferably a “secondary antibody” that is attached to a direct orindirect detectable agent, such a radiolabel or enzyme. Alternatively, a“secondary and tertiary antibody detection system” may be used,including a first detecting antibody that has immunospecificity for thefirst binding region in combination with a second detecting antibodythat has immunospecificity for the first detecting antibody, the seconddetecting antibody being attached to a direct or indirect detectableagent.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the nucleotide and amino acid sequences of the heavy (VH)and light (VL) chain variable region of a scFv form of cloneEJ173/112-C11 (r84/PGN311). ScFv were cloned via Nco/NotI site intopHOG21 (3.7 Kb). The restriction sites used for initial cloning (NcoI,HindIII, MluI and NotI) are italicized and underlined. The linkersequence between VH and VL is in italic.

FIG. 2 shows that clone EJ173/112-C11 (r84/PGN311) scFv binds VEGF. FIG.2 shows the results of an ELISA assay to assess the binding of cloneEJ173/112-C11 (r84), its mother clone and a positive control antibody(murine B9) to plated VEGF-A. Clone EJ173/112-C11 (r84) showed thehighest binding signal and hence the highest affinity.

FIG. 3 shows that clone EJ173/112-C11 (r84/PGN311) scFv effectivelycompetes with the 2C3 antibody for binding to VEGF, which is shown bythe results of a competition ELISA assay. As clone EJ173/112-C11 (r84)effectively competes with the 2C3 antibody for binding to VEGF, thisshows that clone EJ173/112-C11 (r84) binds to substantially the sameepitope as the murine 2C3 anti-VEGF antibody.

FIG. 4 shows that clone EJ173/112-C11 (r84/PGN311) scFv binds to bothmurine VEGF and human VEGF.

FIG. 5 shows the results of a Biacore assay used to assess the bindingaffinity of various scFv antibodies to immobilized VEGF-A. The bindingcurves are shown in FIG. 5 where it can be seen that the scFv form ofEJ173/112-C11 (r84/PGN311) has a noticeably higher binding affinity thanthe single chain form of the mother clone (m). Other curves shown arelabelled v41, r68, r3 and r26.

FIG. 6 shows that EJ173/112-C11 (r84/PGN311) IgG inhibits VEGF-mediatedintracellular cell signalling via VEGFR2, which is shown by the resultsof an in vivo cell assay wherein it is shown that EJ173/112-C11 (r84)IgG inhibits phosphorylation of Erk1/2.

FIG. 7 shows that clone EJ173/112-C11 (r84/PGN311) recognizes thetruncated 121 isoform of VEGF (VEGF121), which is shown by results froman ELISA assay.

FIG. 8A and FIG. 8B together show that r84/PGN311 substantially blocksthe interaction of VEGF with VEGFR2 but does not substantially block theinteraction of VEGF with VEGFR1. VEGF-biotin in the presence or absenceof the indicated antibodies was incubated in wells of an ELISA platethat were coated with soluble VEGFR1 (FIG. 8A) or VEGFR2 (FIG. 8B). Thesignal of VEGF alone (VEGF) or VEGF in the presence of the indicatedantibody was normalized to VEGF alone (100%). The mean+/−SEM is shown.N=12 (4 identical plates with each treatment performed in triplicate). Asignal of less than 50% is considered significant and substantialinhibition of binding. Synagis is a human anti-RSV antibody used as anegative control. For comparison, results with the Avastin (bevacizumab)(Presta et al., 1997) antibody are also presented, which show thatAvastin substantially blocks the interaction of VEGF with both VEGFR2and VEGFR1.

FIG. 9A and FIG. 9B show the scFv expression vector. FIG. 9A shows thescFv expression vector pHOG21. ApR, Ampicillin resistance gene; ColE1,origin of DNA replication; fIIG, intergenic region of phage fl; c-myc,epitope recognized by the monoclonal antibody 9E10; His6, six histidineresidues; pelB, signal peptide of bacterial pectate lyase; P/O, wildtype lac promoter operator. FIG. 9B shows the nucleotide (SEQ ID NO:28)and amino acid (SEQ ID NO:29) sequences of the C-terminal coding region.

FIG. 10A, FIG. 10B and FIG. 10C show that tumor associated macrophagesexpress VEGFR2. FIG. 10A shows co-localization of T014 (VEGFR2 antibody)and F4/80 (macrophage marker) staining on tumor sections from controltreated or 2C3 treated animals. FIG. 10A shows that 2C3 decreasesmacrophage infiltration. However, both the control and 2C3 groupsdemonstrate co-localization of VEGFR2 and macrophage markers. FIG. 10Bshows the number of cells double positive for one of three differentmacrophage markers and VEGFR2. FIG. 10C uses two different antibodies toVEGFR2 to show that peritoneal macrophages from tumor bearing animalsexpress VEGFR2.

FIG. 11 shows that r84/PGN311 inhibits the growth of MDA-MB-231 tumors.FIG. 11 shows results from a study using an in vivo (mouse) MDA-MB-231breast cancer tumor model and the effect of r84, Avastin or saline(control) on tumor volume. Mean tumor volume+/−SEM is shown. Avastin andr84 treated mice have tumor volumes that are significantly smaller thancontrol treated animals.

FIG. 12 shows results from the same study as shown in FIG. 11, exceptthat FIG. 12 shows the tumor weight/body weight ratio for individualanimals in each group. Avastin and r84/PGN311 treated mice have tumorweight/body weight ratios that are significantly smaller than controltreated animals.

FIG. 13 shows that r84/PGN311 inhibits the growth of A673 tumors. FIG.13 shows results from a study using an in vivo (mouse) A673 tumor modeland the effect of r84, 2C3 or a control antibody (Synagis—humananti-RSV) on tumor volume. Mean tumor volume+/−SEM is shown. 2C3 and r84treated mice have tumor volumes that are significantly smaller thancontrol treated animals. 2C3 and r84 are thus effective at controllingthe growth of A673 tumors.

FIG. 14 shows that r84/PGN311 significantly reduces infiltration oftumor associated macrophages. Tumors were taken from mice withMDA-MB-231 tumor cells and sectioned and stained with antibodies to amacrophage marker (Mac-3). Three tumors from control animals and threetumors each from r84 and 2C3 treated animals were analyzed and 5 imagesfrom each tumor were studied. FIG. 14 shows that tumors from r84 and 2C3treated animals showed significantly reduced expression of themacrophage marker Mac-3, and that r84 has a more pronounced effect than2C3 (p<0.01 for r84).

FIG. 15 shows that r84/PGN311 significantly reduces microvessel densityin MDA-MB-231 animal model tumors. Tumors were taken from mice withMDA-MB-231 tumor cells and sectioned and stained with antibodies tomouse endothelial cells (MECA-32). Three tumors from control animals andthree tumors each from r84 and 2C3 treated animals were analyzed and 5images from each tumor were studied. FIG. 15 shows that tumors from r84and 2C3 treated animals showed a significantly reduced number of bloodvessels/high power field (MECA-32, p<0.0001).

FIG. 16A and FIG. 16B show that r84/PGN311 selectively blocks the VEGFR2pathway. FIG. 16A shows that r84/PGN311 inhibits the VEGF stimulated(+VEGF) phosphorylation of Erk1/2 (pERK1/2) and PLC-γ (pPLC-γ) on VEGFR2expressing cells (HDMEC). The positive control Avastin also inhibits theVEGF stimulated phosphorylation of Erk1/2 and PLC-γ. FIG. 16B shows thatr84/PGN311 does not inhibit the VEGF stimulated phosphorylation ofVEGFR1 on VEGFR1 expressing cells (PAE Flt) whereas the positivecontrol, Avastin, does inhibit phosphorylation of VEGFR1.

FIG. 17A, FIG. 17B, FIG. 17C and FIG. 17D show that r84/PGN311 leads toa significant reduction in growth of tumors produced by non-small celllung cancer cell lines. FIG. 17A, FIG. 17B, FIG. 17C and FIG. 17D showresults from studies using an in vivo mouse model and four differentnon-small cell lung cancer cell lines, H460 (FIG. 17A), H1299 (FIG.17B), H358 (FIG. 17C) and A549 (FIG. 17D). The effect of r84/PGN311,Avastin or a control antibody (Synagis or XTL) on tumor weight is shown(mean weight of tumors+/−SEM is shown). r84/PGN311- and Avastin-treatedmice have mean tumor weights that are significantly lower than controltreated animals. r84/PGN311 and Avastin are thus effective atcontrolling the growth of non-small cell lung cancer cell lines.r84/PGN311 performs better than Avastin at least in the H460 (FIG. 17A),H1299 (FIG. 17B) and the A549 (FIG. 17D) models. r84 is significantlybetter than Avastin in the A549 model (FIG. 17D).

FIG. 18A, FIG. 18B and FIG. 18C show that the lymphatic vessel densityin r84-treated tumors is significantly lower than in control tumors.FIG. 18A (six panels) is immunofluorescence staining of frozenMDA-MB-231 tumor sections showing the lymphatic markers, podoplanin(green), Prox1 (red) and the merged images, in control (top panels) andr84-treated tumors (bottom panels). FIG. 18B (two panels) showsMDA-MB-231 tumor sections stained for LYVE-1 in control (top panel) andr84-treated tumors (bottom panel). The pattern of LYVE-1 staining (FIG.18B) is similar to that for podoplanin and Prox1 (FIG. 18A). The entirearea of each LYVE-1 stained tumor section was examined at lowmagnification and the percent of LYVE-1 positive area was determined foreach field using NIS-Elements imaging software (FIG. 18C). The tenfields with the highest LYVE-1 positive percent area were averagedtogether to yield a final score for each tumor and group means weretested for significance by an unpaired student's t-test. The percent ofLYVE-1 positive area of control tumors (7.03±1.013; n=6) wassignificantly greater than r84 treated tumors (2.23±0.986; n=5), withP=0.0042.

FIG. 19 shows that r84 in fully human and murine chimeric IgG formatsbinds to both murine VEGF and human VEGF. Human VEGF (0.5 μg/mL, R&D) ormouse VEGF (0.5 μg/mL, Sigma) was coated onto the bottom of 96-wellplates. Wells were blocked and then incubated with the indicatedconcentration of human r84 (blue lines) or mouse chimeric r84 (greenlines). Antibody bound to the wells was detected by incubation withanti-human Fc or anti-mouse HRP-conjugated antibody. Average absorbanceis displayed.

FIG. 20A and FIG. 20B show that r84/PGN311 potently inhibitsVEGF-induced migration of VEGFR2-expressing endothelial cells. HDMEC(FIG. 20A) and PAE KDR-expressing cells (FIG. 20B) were used intranswell assays. The cells were either not stimulated (NS), or exposedto VEGF at 100 ng/ml to stimulate migration (VEGF), and the ability of a500-fold molar excess of r84, Avastin (Avas) or control (Cntl)antibodies (IgG format) to inhibit VEGF-induced migration was tested.VEGF induces migration in comparison to not stimulated cells (p<0.01).r84 and Avastin inhibit VEGF-induced migration (***, p<0.0001 vs. VEGFalone).

FIG. 21 shows that r84/PGN311 does not inhibit VEGF-induced migration ofVEGFR1-expressing endothelial cells. PAE Flt1-expressing cells wereeither not stimulated (NS), or exposed to VEGF (VEGF) to stimulatemigration (VEGF), and the ability of r84, Avastin (Avas) or control(Cntl) antibodies to inhibit VEGF-induced migration was tested. VEGFinduces migration in comparison to not stimulated cells. Avastinsignificantly inhibits VEGF-induced migration, whereas r84 does not.Thus, FIG. 21 shows that r84/PGN311 does not inhibit VEGF-inducedmigration of VEGFR1-expressing endothelial cells. PAE Flt1, endothelialcells that express VEGFR1 exclusively, were serum-starved for 24 hoursand then plated in serum-free media in transwell inserts (8 μM pores,5,000 cells/insert). Migration to the underside of the membrane wasstimulated by adding the following to the well below the insert:serum-free media (NS); VEGF (100 ng/ml); VEGF+a control IgG (Cntl);VEGF+Avastin (Avastin); VEGF+r84 (r84). The cells were allowed tomigrate for 24 hours at which time the membranes were removed, cellsremoved from the upper surface of the membrane, fixed, and stained withDAPI. DAPI stained nuclei on the underside of the member were thencounted by fluorescence microscopy and quantified using software(Elements, Nikon). *, p<0.05 r84 vs Avastin; **, p<0.01 Avastin vscontrol.

FIG. 22 shows that r84/PGN311 markedly reduces the growth of Panelpancreatic tumor cells in mice. Mice bearing Panel pancreaticadenocarcinoma cells were given either r84/PGN311 IgG or Synagis(negative control). Tumor volumes are depicted over the time course oftreatment. Thus, FIG. 22 shows that r84/PGN311 reduces the growth ofsubcutaneous human pancreatic tumor xenografts. Panel tumor cells wereinjected subcutaneously into SCID mice (2×10⁶ cells/animal) on day 0.Mice were treated starting on Day 1 2×/week with 500 μg of a control IgG(Synagis) or r84. Tumor volume was monitored over time using calipers.Mean (SEM) tumor volume (n=5/group) versus day post tumor cell injection(TCI) is shown.

FIG. 23 shows that the mouse chimeric version of r84/PGN311 prolongssurvival of mice bearing syngeneic 4T1 mammary tumors. Murine 4T1 tumorswere injected orthotopically into Balb/C mice (n=8 mice per group).Either the mouse chimeric version of r84/PGN311 (mcr84, red line) orcontrol (Control, black line) antibody was administered via i.p.injection twice a week starting on day 12 and continuing for 3 weeks.r84/PGN311 prolonged survival in comparison to control.

FIG. 24 shows the level of mouse VEGF in sera from tumor-bearing micethat were treated with control IgG, avastin, 2C3 or r84 (as indicated).The sera was collected and assayed by ELISA for the level of mouse VEGFusing a kit from R&D systems. In addition, an aliquot of sera from r84treated mice was pre-cleared with Protein G beads. Supernatant from theProtein G cleared sera (r84 supe) was also tested.

FIG. 25 shows that r84, and to a lesser extent Avastin (bev), decreasesthe infiltration of CD11b+/Gr1+ cells into MDA-MB-231 tumors in vivo,while 2C3 does not. The reduction for r84 is 39%. One-way ANOVAindicates that the decreased infiltration observed with r84 treatedanimals, but not the 2C3 or Avastin (bev) treated animals, isstatistically different from the control treated animals (designated **,p<0.01)

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Solid tumors and carcinomas account for more than 90% of all cancers inman. Although the use of monoclonal antibodies and immunotoxins has beeninvestigated in the therapy of lymphomas and leukemias, these agentshave been disappointingly ineffective in clinical trials againstcarcinomas and other solid tumors (Abrams and Oldham, 1985). A principalreason for the ineffectiveness of antibody-based treatments is thatmacromolecules are not readily transported into solid tumors. Even oncewithin a tumor mass, these molecules fail to distribute evenly due tothe presence of tight junctions between tumor cells, fibrous stroma,interstitial pressure gradients and binding site barriers (Dvorak etal., 1991a). In developing new strategies for treating solid tumors, themethods that involve targeting the vasculature of the tumor, rather thanthe tumor cells, offer distinct advantages. An effective destruction orblockade of the tumor vessels arrests blood flow through the tumor andresults in an avalanche of tumor cell death. Antibody-toxin andantibody-coagulant constructs have already been effectively used in thespecific targeting and destruction of tumor vessels, resulting in tumornecrosis (Burrows et al., 1992; Burrows and Thorpe, 1993; WO93/17715;WO96/01653; U.S. Pat. Nos. 5,855,866; 5,877,289; 5,965,132; 6,051,230;6,004,555; 6,093,399.

Where antibodies, growth factors or other binding ligands are used tospecifically deliver a coagulant to the tumor vasculature, such agentsare termed “coaguligands”. A currently preferred coagulant for use incoaguligands is truncated Tissue Factor (tTF) (Huang et al., 1997;WO96/01653; U.S. Pat. No. 5,877,289). TF is the major initiator of bloodcoagulation. At sites of injury, Factor VII/VIIa in the blood comes intocontact with, and binds to, TF on cells in the perivascular tissues. TheTF:VIIa complex, in the presence of the phospholipid surface, activatesfactors IX and X. This, in turn, leads to the formation of thrombin andfibrin and, ultimately, a blood clot.

A range of suitable target molecules that are available on tumorendothelium, but largely absent from normal endothelium, have beendescribed. For example, expressed targets may be utilized, such asendoglin, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a TIE, a ligandreactive with LAM-1, a VEGF/VPF receptor, an FGF receptor, α_(v)β₃integrin, pleiotropin or endosialin (U.S. Pat. Nos. 5,855,866; 5,877,289and 6,004,555; Burrows et al., 1992; Burrows and Thorpe, 1993; Huang etal., 1997; each incorporated herein by reference).

Other targets inducible by the natural tumor environment or followingintervention by man are also targetable entities, as described in U.S.Pat. Nos. 5,776,427 and 6,036,955; each incorporated herein byreference). When used in conjunction with prior suppression in normaltissues and tumor vascular induction, MHC Class II antigens may also beemployed as targets (U.S. Pat. Nos. 5,776,427; 6,004,554 and 6,036,955;each incorporated herein by reference).

Adsorbed targets are another suitable group, such as VEGF, FGF, TGFβ,HGF, PF4, PDGF, TIMP, a ligand that binds to a TIE or a tumor-associatedfibronectin isoform (U.S. Pat. Nos. 5,877,289 and 5,965,132; eachincorporated herein by reference). Fibronectin isoforms are ligands thatbind to the integrin family of receptors. Tumor-associated fibronectinisoforms are targetable components of both tumor vasculature and tumorstroma.

One currently preferred marker for such clinical targeting applicationsis receptor-associated VEGF. In fact, assemblies of VEGF:receptorcomplexes are one of the most specific markers of tumor vasculatureobserved to date (U.S. Pat. Nos. 5,877,289; 5,965,132 and 6,051,230;Lin-Ke et al., 1996; Dvorak et al., 1991b).

The VEGF:receptor complex presents an attractive target for the specificdelivery of drugs or other effectors to tumor endothelium—as tumors arerich in cytokines and growth factors and as VEGF receptors areupregulated under the hypoxic conditions that are found in most solidtumors (Mazure et al., 1996; Forsythe et al., 1996; Waltenberger et al.,1996; Gerber et al., 1997; Kremer et al., 1997). Upregulation of boththe ligand and its receptor specifically in the tumor microenvironmentleads to a high concentration of occupied receptor on tumor vascularendothelium, as compared with the endothelium in normal tissue (U.S.Pat. Nos. 5,877,289 and 5,965,132). Dvorak and colleagues also showedthat rabbit polyclonal antibodies directed against the N-terminus ofVEGF selectively stain tumor blood vessels after injection into micebearing syngeneic tumors (Lin-Ke et al., 1996).

The role of VEGF as a target for clinical intervention is not limited toimmunotoxin or coaguligand therapies. Indeed, VEGF is one of the keyfactors involved in angiogenesis of solid tumors (Ferrara, 1995; Potgenset al., 1995), being both a potent permeability agent (Senger et al.,1983; Senger et al., 1990; Senger et al., 1986) and endothelial cellmitogen (Keck et al., 1989; Connolly et al., 1989; Thomas, 1996). Thelink between VEGF and angiogenesis has led to proposals of varioustherapeutic strategies aimed at VEGF intervention (Siemeister et al.,1998).

A. VEGF and VEGF Receptors

Vascular endothelial growth factor isoform A (VEGF-A, succinctly termed“VEGF” in the present application) is a multifunctional cytokine that isinduced by hypoxia and oncogenic mutations. VEGF is a primary stimulantof the development and maintenance of a vascular network inembryogenesis. It functions as a potent permeability-inducing agent, anendothelial cell chemotactic agent, an endothelial survival factor, andendothelial cell proliferation factor (Thomas, 1996; Neufeld et al.,1999). Its activity is required for normal embryonic development (Fonget al., 1995; Shalaby et al., 1995), as targeted disruption of one orboth alleles of VEGF results in embryonic lethality (Carmeliet et al.,1996; Ferrara et al., 1996).

VEGF is an important factor driving angiogenesis or vasculogenesis innumerous physiological and pathological processes, including woundhealing (Frank et al., 1995; Burke et al., 1995), diabetic retinopathy(Alon et al., 1995; Malecaze et al., 1994), psoriasis (Detmar et al.,1994), atherosclerosis (Inoue et al., 1998), rheumatoid arthritis(Harada et al., 1998; Nagashima et al., 1999), solid tumor growth (Plateet al., 1994; Claffey et al., 1996).

A wide variety of cells and tissues produce VEGF, which exists in atleast five isoforms (121, 145, 165, 189, and 206 amino acids) that aresplice variants encoded by the same gene (Houck et al., 1991; Ferrara etal., 1991; Tischer et al., 1991). The two smaller isoforms, 121 and 165,are secreted from cells (Houck et al., 1991; Anthony et al., 1994).Secreted VEGF is an obligate dimer of between 38-46 kDa in which themonomers are linked by two disulfide bonds.

VEGF dimers bind with high affinity to two well-characterized receptors,VEGFR1 (FLT-1) and VEGFR2 (KDR/Flk-1), which are selectively expressedon endothelial cells (Flt-1 and Flk-1 are the mouse homologues). TheK_(d) of VEGF binding to VEGFR1 and VEGFR2 is 15-100 pM and 400-800 pM,respectively (Terman et al., 1994). A recently identified third cellsurface protein, neuropilin-1, also binds VEGF with high affinity(Olander et al., 1991; De Vries et al., 1992; Terman et al., 1992; Sokeret al., 1998).

VEGFR1 and VEGFR2 are members of the Type III receptor tyrosine kinase(RTK III) family that is characterized by seven extracellular IgG-likerepeats, a single spanning transmembrane domain, and an intracellularsplit tyrosine kinase domain (Mustonen and Alitalo, 1995). Until veryrecently, VEGFR1 and VEGFR2 were thought to be almost exclusivelyexpressed on endothelial cells (Mustonen and Alitalo, 1995). AlthoughVEGFR1 and VEGFR2 have been reported to have different functions withrespect to stimulating endothelial cell proliferation, migration, anddifferentiation (Waltenberger et al., 1994; Guo et al., 1995), theprecise role that each receptor plays in VEGF biology and endothelialcell homeostasis was not clearly defined prior to the present invention.

Recent studies using knockout mice have shown each of VEGF, VEGFR1 andVEGFR2 to be essential for vasculogenesis, angiogenesis and embryodevelopment (Fong et al., 1995; Shalaby et al., 1995; Hiratsuka et al.,1998). In studies of lethal knockouts, the phenotypes associated withthe lack of each receptor were different. Targeted disruption of VEGFR2resulted in an embryo that lacked endothelial cell differentiation andfailed to form yolk sac blood islands or go through vasculogenesis(Shalaby et al., 1995). VEGFR1 null mutants showed impairedvasculogenesis, disorganized assembly of endothelial cells, and dilatedblood vessels (Fong et al., 1995; Hiratsuka et al., 1998). VEGFR1evidently has a vital biological role.

VEGFR1 has a higher affinity for VEGF than VEGFR2, although it has alower tyrosine kinase activity. This suggests that the extracellulardomain of VEGFR1 is particularly important. This hypothesis was stronglysupported by results from studies in knockout mice in which the tyrosinekinase domain of VEGFR1 was deleted whilst leaving the VEGF bindingdomain intact (Hiratsuka et al., 1998). The VEGFR1-tyrosine kinasedeficient embryos developed normal blood vessels and survived to term(Hiratsuka et al., 1998).

In addition to the earlier knockouts (Fong et al., 1995; Shalaby et al.,1995), the Hiratsuka et al. (1998) studies indicate that VEGFR1 has avital biological role. However, tyrosine kinase signaling does not seemto be the critical factor. It is interesting to note that macrophagesfrom the VEGFR1 knockout mice did not exhibit VEGF-induced chemotaxis(Hiratsuka et al., 1998; incorporated herein by reference), therebyimplicating VEGFR1 as the receptor responsible for mediating thisimportant biological response to VEGF.

Certain groups have reported VEGFR2 to be the dominant signalingreceptor in VEGF-induced mitogenesis, and permeability (Waltenberger etal., 1994; Zachary, 1998; Korpelainen and Alitalo, 1998). The role ofVEGFR1 in endothelial cell function is much less clear, althoughfunctions in macrophage migration and chemotaxis were documented in theHiratsuka et al. (1998) studies discussed above.

Clauss et al. (1996; incorporated herein by reference) also reportedthat VEGFR1 has important roles in monocyte activation and chemotaxis.In fact, cells of the macrophage/monocyte lineage express only VEGFR1,which is the receptor responsible for mediating monocyte recruitment andprocoagulant activity (Clauss et al., 1996). VEGF binding to VEGFR1 onmonocytes and macrophages also acts by raising intracellular calcium andinducing tyrosine phosphorylation (Clauss et al., 1996).

Binding of the VEGF dimer to the VEGF receptor is believed to inducereceptor dimerization. Dimerization of the receptor then causesautotransphosphorylation of specific tyrosine residues, Y801 and Y1175,and Y1213 and Y1333 on the intracellular side of VEGFR2 and VEGFR1,respectively. This leads to a signal transduction cascade, whichincludes activation of phospholipase Cγ (PLCγ) and phosphatidylinositol3-kinase (PI3K) and an increase in intracellular calcium ions (Hood andMeininger, 1998; Hood et al., 1998; Kroll and Waltenberger, 1998).

The intracellular events further downstream in VEGF-induced signalingare less clear, although a number of groups have shown that nitric oxide(NO) is produced after VEGF activation of VEGFR2 (Hood and Meininger,1998; Hood et al., 1998; Kroll and Waltenberger, 1998). Activation ofVEGFR2, but not VEGFR1, by VEGF has also been shown to activate Src andthe Ras-MAP kinase cascade, including the MAP kinases, ERK1 and ERK2(Waltenberger et al., 1994, 1996; Kroll and Waltenberger, 1997).

The role of VEGFR1 in endothelial cell function is much less clear,particularly as Flt-1 tyrosine kinase-deficient mice are viable anddevelop normal vessels (Hiratsuka et al., 1998). It has been suggestedthat the main biological role of VEGFR1 on endothelial is as anon-signaling ligand-binding molecule, or “decoy” receptor that might berequired to present VEGF to VEGFR2.

The connection between VEGF and pathological angiogenic conditions hasprompted various attempts to block VEGF activity. These include thedevelopment of certain neutralizing antibodies against VEGF (Kim et al.,1992; Presta et al., 1997; Sioussat et al., 1993; Kondo et al., 1993;Asano et al., 1995). Antibodies against VEGF receptors have also beendescribed, such as described in U.S. Pat. Nos. 5,840,301 and 5,874,542and, subsequent to the present invention, in WO 99/40118. U.S. Pat. Nos.5,840,301 and 5,874,542 indeed suggest that blocking VEGF receptorsrather than VEGF itself is advantageous for various reasons.

Soluble receptor constructs (Kendall and Thomas, 1993; Aiello et al.,1995; Lin et al., 1998; Millauer et al., 1996), tyrosine kinaseinhibitors (Siemeister et al., 1998), antisense strategies, RNA aptamersand ribozymes against VEGF or VEGF receptors have also been reported(Saleh et al., 1996; Cheng et al., 1996; each incorporated herein byreference).

B. Anti-VEGF Antibodies

B1. Antibody Properties

The application of various inhibitory methods has been shown to be atleast somewhat effective in either blocking angiogenesis and/orsuppressing tumor growth by interfering with VEGF signaling. In fact,monoclonal antibodies against VEGF have been shown to inhibit humantumor xenograft growth and ascites formation in mice (Kim et al., 1993;Asano et al., 1995; 1998; Mesiano et al., 1998; Luo et al., 1998a;1998b; Borgstrom et al., 1996; 1998).

The antibody A4.6.1 is a high affinity anti-VEGF antibody capable ofblocking VEGF binding to both VEGFR1 and VEGFR2 (Kim et al., 1992;Wiesmann et al., 1997; Muller et al., 1998). Alanine scanningmutagenesis and X-ray crystallography of VEGF bound by the Fab fragmentof A4.6.1 showed that the epitope on VEGF that A4.6.1 binds is centeredaround amino acids 89-94. This structural data demonstrates that A4.6.1competitively inhibits VEGF from binding to VEGFR2, but inhibits VEGFfrom binding to VEGFR1 most likely by steric hindrance (Muller et al.,1998; Keyt et al., 1996; each incorporated herein by reference)

A4.6.1 is the most extensively utilized neutralizing anti-VEGF antibodyin the literature to date. It has been shown to inhibit the growth andVEGF-induced vascular permeability of a variety of human tumors in mice(Brem, 1998; Baca et al., 1997; Presta et al., 1997; Mordenti et al.,1999; Borgstrom et al., 1999; Ryan et al., 1999; Lin et al., 1999; eachspecifically incorporated herein by reference). A4.6.1 also inhibitsascites formation in a well-characterized human ovarian carcinoma mousemodel and tumor dissemination in a metastasis mouse model. A4.6.1 hasbeen humanized by monovalent phage display techniques (Brem, 1998; Bacaet al., 1997; Presta et al., 1997; each incorporated herein byreference). The resulting humanized antibody, termed Avastin(bevacizumab), has been approved for clinical use (Hurwitz et al.,2004).

Despite success in the art with neutralizing antibodies against VEGF,the present inventors realized that new antibodies, particularly humanantibodies with a more precisely defined mode of interaction with VEGFR1(FLT-1) and/or VEGFR2 (KDR/Flk-1) would of benefit for a variety ofreasons. For example, the development of anti-VEGF antibodies thatselectively block the interaction of VEGF with only one of the two VEGFreceptors would allow for a more precise dissection of the pathwaysactivated by VEGF in cells that express both VEGFR1 and VEGFR2.

The present inventors believed that human antibodies of definedepitope-specificity that blocked VEGF binding to only one receptor(VEGFR2s) will have clinical benefits. The knockout mice studies ofHiratsuka et al. (1998) show that both VEGFR1 and VEGFR2 have importantbiological roles. Prior to the present invention, realisticopportunities for therapeutic intervention aimed at inhibitingVEGF-mediated effects through only one of the two receptors werehampered by the lack of effective, tailored inhibitory agents optimizedfor human administration.

Given the need for therapeutic specific human antibodies that blockangiogenesis, human antibodies have been identified that are reactiveagainst an epitope on VEGF that specifically and substantially blocksits interaction with VEGF receptor 2 (VEGFR2, KDR/Flk-1), but does notsubstantially blocks its interaction with VEGF receptor 1 (VEGFR1,Flt-1).

The present inventors first developed a range of fully human anti-VEGFantibodies that competed with the murine antibody 2C3 for binding toVEGF. A number of antibody clones displaying high affinity for VEGF andshowing selective disruption for the interaction between VEGF and VEGFR2and not between VEGF and VEGFR1 were selected for further analysis.Eventually one of these clones, termed a “mother clone”, was subjectedto maturation, after which a new clone was selected that displayedfurther important and significant improvements, for example, a betterbinding affinity to both mouse VEGF and human VEGF, a higher stabilityin serum and a reduced tendency to form aggregates in scFv format. Thisantibody is called r84 (and PGN311) and displays excellent bindingaffinity to VEGF, with a Kd in IgG format in the order of 7 nM or less,which is well within the range shown to be effective in human therapy.

Furthermore, the r84 antibody is shown herein to significantly reducetumor volume/tumor growth in several art-accepted in vivo tumor models(specifically, the A673 rhabdomyosarcoma tumor model, the MDA-MB 231breast cancer cell tumor model, various human non-small cell lung cancermodels, Panc 1 pancreatic cancer cell tumor model and 4T1 mammary tumormodel). Notably, the results with r84 are at least as good as thehumanized anti-VEGF antibody termed Avastin, which has been approved forclinical use. A fully human antibody such as r84 will provide advantagesover the available humanized antibody. In addition, r84 has theadvantageous property of binding to mouse VEGF and human VEGF. Theability to bind mouse VEGF is an important advantage that the r84antibody displays over 2C3 and Avastin. Furthermore, results from theMDA-MB 231 tumor model also show that r84 significantly reducesinfiltration of tumor associated macrophages, which are now known toplay a positive role in cancer development and metastasis and thus to bedetrimental to patients In this regard, it has been shown that r84significantly reduces expression of the macrophage marker Mac-3(p<0.01). In addition, results from the MDA-MB 231 tumor model show thatr84 significantly (p<0.0001) reduces the number of blood vessels intumors and hence significantly reduces microvessel density (MVD) intumors.

r84 has also been shown to significantly inhibit VEGF induced migrationof VEGFR2 expressing cells and to significantly reduce lymphatic vesseldensity in MDAMB231 tumors. The effect on lymphatic vessel densitysupports the use of the human antibodies of the invention to inhibitlymphangiogenesis.

A further advantageous property shown by r84 is the ability tosignificantly reduce infiltration of myeloid-derived suppressor cells,in particular CD11b+/Gr1+ cells, into tumors. Furthermore, this propertyis not shown by the 2C3 antibody and only at a more reduced level byAvastin. Thus, further studies in MDA-MB-231 tumor-bearing mice haveshown that significantly less CD11b/Gr1 double positive cells infiltratetumors in r84-treated animals as opposed to control. In comparativestudies, neither the 2C3 antibody nor Avastin showed a statisticallysignificant decrease in CD11b+/Gr1+ infiltration, although somereduction was measurable in Avastin-treated animals. The reduction inthe number of double positive cells observed in r84/PGN311 treatedanimals is 39% (FIG. 25).

The reduced infiltration of myeloid derived suppressor cells CD11b+/Gr1+is of special interest, as cells expressing both markers have recentlybeen associated with mediation of tumor refractoriness to anti-VEGFtherapy (Shojaei et al., 2007). Myeloid-derived suppressor cells(CD11b+Gr1+) are also an important contributor to tumor progression. Inthe tumor microenvironment these cells secrete immunosuppressivemediators and induce T-lymphocyte dysfunction (Gabrilovich et al., 2001;Serafini et al., 2004).

As CD11b+/Gr1+ cells are associated with tumor refractoriness toanti-VEGF therapy and contribute to tumor progression, the effect ofr84/PGN311 to reduce infiltration or recruitment of these cells intotumors clearly has a potential importance for therapeutic applicationsof r84, in particular therapeutic applications related to the treatmentof angiogenic diseases, including cancer.

Indeed, as the results herein show that the tumor infiltration ofCD11b+/Gr1+ cells is least pronounced/significantly lower in the animalstreated with r84/PGN311, it suggests that treatment with r84 is likelyto be less prone to the development of drug resistance or refractorinessto anti-VEGF therapy than treatment with other drugs targeting VEGF,e.g. other anti-VEGF antibodies. In addition, given the proposed role ofCD11b+/Gr1+ cells in tumor progression, the ability of r84/PGN311 toreduce infiltration or recruitment of such cells into tumors might wellform part of the mechanism involved in the anti-tumor activity, e.g. theinhibition of tumor growth shown by r84/PGN311.

It has also been shown that chronic administration of r84/PGN311 doesnot induce toxicity in mice.

These are further positive indications of the therapeutic potential ofthe r84 antibody.

B2. VEGFR2-Blocking, Human Anti-VEGF Antibodies

An important part of this invention, confirmed using ELISA, receptorbinding assays and receptor activation assays, is that the antibodies ofthe invention selectively block the interaction of VEGF with VEGFR2(KDR/Flk-1), but not VEGFR1 (FLT-1). The antibodies inhibit VEGF-inducedphosphorylation of VEGFR2 and inhibits signalling via the VEGFR2. Theantibodies also have potent anti-tumor activity, arresting the growth ofestablished human solid tumors in art-accepted animal models of humancancer. In addition, the human antibodies of the invention haveanti-angiogenic properties and reduce microvessel density in tumors.

These properties demonstrate the usefulness of the antibodies indissecting the pathways that are activated by VEGF in cells that expressboth VEGFR1 and VEGFR2, as well as highlighting the importance of VEGFR2activity in the process of tumor growth and survival. More importantly,they provide a unique mode of therapeutic intervention for a humanantibody, allowing specific inhibition of VEGFR2-induced angiogenesis,without concomitant inhibition of VEGFR1-mediated events, such asosteoclast and chondroclast function.

The antibodies of the present invention, succinctly termed“VEGFR2-blocking, human anti-VEGF antibodies”, represent an advance inthe field and provide numerous advantages, both in terms of uses inunconjugated or “naked” form and when conjugated to or associated withother therapeutic agents.

The in vitro binding studies of the present invention demonstrate thatthe human antibodies block the binding of VEGF to VEGFR2, but do notinhibit the binding of VEGF to VEGFR1.

The human antibodies of the present invention are thus significantlyimproved over other blocking antibodies to VEGF, including the murineA4.6.1 antibody and its humanized counterpart, Avastin (bevacizumab).The A4.6.1 and Avastin anti-VEGF antibodies block the binding of VEGF toboth VEGF receptors. Crystallographic and mutagenesis studies have shownthat the binding epitopes for VEGFR2 and VEGFR1 are concentrated towardsthe two symmetrical poles of the VEGF dimer (Wiesmann et al., 1997;Muller et al., 1997). The binding determinants on VEGF that interactwith the two receptors overlap partially and are distributed over fourdifferent segments that span across the dimer surface (Muller et al.,1998). Antibody 4.6.1 binds to a region of VEGF within the receptorbinding region of both receptors (Muller et al., 1998).

Studies on the effect of the human antibodies of the invention onVEGF-induced phosphorylation of the receptors showed that the antibodiesdo block VEGF-induced phosphorylation of VEGFR2. Studies have also shownthat the human antibodies of the invention inhibit cell signalling viaVEGFR2, for example, the antibodies have been shown to inhibitphosphorylation of Erk 1/2 and PLC-γ in in vitro assays.

The human antibodies of the invention inhibit the growth of human tumortypes in vivo. The magnitude of tumor growth suppression by the humanantibodies of the invention is similar to that using differentneutralizing anti-VEGF antibodies, including Avastin. The effectivenessof these human antibodies, being similar to what other investigatorshave found using different anti-VEGF antibodies, further demonstratesthe role of VEGF in tumor angiogenesis and tumor growth. However, thehuman antibodies of the invention should provide a safer therapeutic,based on the specific inhibitory properties discussed herein and inlight of being fully human antibodies.

The fact that regressions, rather than tumor stasis, can be achievedsuggests that VEGF is providing more than just an angiogenic signal fortumor endothelium. Benjamin et al. (1999) recently reported that tumorscontain a large fraction of immature blood vessels that have yet toestablish contact with periendothelial cells and that these bloodvessels are dependent upon VEGF for survival. It is possible thatneutralization of VEGF causes these immature blood vessels to undergoapoptosis, thereby reducing the existing vascular network in the tumor.It is also possible that a dynamic process of vascular remodeling occursin tumors, involving both vessel formation and vessel regression, andthat neutralization of VEGF prevents vessel formation leading to a netshift towards vessel regression.

The finding that the human antibodies of the invention suppress tumorgrowth as completely as Avastin (if not more so) indicates a dominantrole for VEGFR2 in tumor angiogenesis. The multistep process ofangiogenesis requires endothelial cell chemotaxis, metalloproteinaseproduction, invasion, proliferation and differentiation. VEGFR1 may haveno role in these processes, or may assist in the processes by bindingVEGF and presenting it to the signaling receptor, VEGFR2.

The comparable figures for the human antibodies of the invention andAvastin in tumor treatment are highly relevant: the human antibodies ofthe invention are at least as effective as Avastin, although they onlyinhibit VEGF binding to VEGFR2 and not VEGFR1. The present studiestherefore indicate that VEGFR1 does not play a notable role inVEGF-mediated tumor angiogenesis, and further suggest that VEGFR1specific inhibitors may not influence tumor angiogenesis. These resultsalso signify that the human antibodies of the invention can be equallyor more effective than Avastin, whilst causing less side-effects.

The ability to specifically block VEGF binding to and activation ofVEGFR2, but not VEGFR1 (Flt-1), has clinical importance. The humanantibodies of the present invention thus block VEGF angiogenic activity,but do not inhibit other beneficial actions of VEGF, mediated throughVEGFR1, such as those on certain immune and bone cells. One area ofclinical importance thus concerns the ability of the human antibodies ofthis invention to function in vivo without inhibiting the beneficialeffects of osteoclasts and chondroclasts. This means that use of thepresent VEGFR2-blocking, human anti-VEGF antibody therapeutics will notbe associated with side effects on bone and/or cartilage.

In vivo studies have shown that VEGF couples hypertrophic cartilageremodeling, ossification and angiogenesis during endochondral boneformation and that VEGF is essential for cartilage remodeling (Gerber etal., 1999; specifically incorporated herein by reference). Inactivationof VEGF signaling through VEGFR1, by administration of the solubleVEGFR1 receptor chimeric protein (Flt-(1-3)-IgG), was shown to impairtrabecular bone formation and the expansion of the hypertrophicchondrocyte zone by decreasing the recruitment and/or differentiation ofchondroclasts (Gerber et al., 1999).

It has further been shown that VEGF can substitute for macrophagecolony-stimulating factor (M-CSF) in the support of osteoclast functionin vivo (Niida et al., 1999; specifically incorporated herein byreference). In studies using osteopetrotic (op/op) mice with adeficiency in osteoclasts resulting from a mutation in the M-CSF gene,injection of recombinant human M-CSF (rhM-CSF) allows osteoclastrecruitment and survival. In recent studies, it was shown that a singleinjection of recombinant human VEGF can similarly induce osteoclastrecruitment in op/op mice (Niida et al., 1999).

Niida et al. (1999) reported that as osteoclasts predominantly expressVEGFR1, and the activity of recombinant human placenta growth factor 1on osteoclast recruitment was comparable to that of rhVEGF, thebeneficial effects of VEGF signaling in osteopetrotic (op/op) mice aremediated via the VEGF receptor 1 (VEGFR-1). These authors further showedthat rhM-CSF-induced osteoclasts died after VEGF was inhibited (using aVEGFR1 receptor chimeric protein, VEGFR1/Fc), but that such effects wereabrogated by concomitant injections of rhM-CSF. Osteoclasts supported byrhM-CSF or endogenous VEGF showed no significant difference in in vivoactivity (Niida et al., 1999).

Mutant op/op mice undergo an age-related resolution of osteopetrosisaccompanied by an increase in osteoclast number. In the Niida et al.(1999) studies, most of the osteoclasts disappeared after injections ofanti-VEGF antibody, demonstrating that endogenously produced VEGF isresponsible for the appearance of osteoclasts in the mutant mice. Inaddition, rhVEGF replaced rhM-CSF in the support of in vitro osteoclastdifferentiation. These results demonstrate that M-CSF and VEGF haveoverlapping functions in the support of osteoclast function and thatVEGF acts through the VEGFR-1 receptor (Niida et al., 1999).

It can thus be concluded that the VEGFR2-blocking, human anti-VEGFantibodies of the invention do not block VEGF from binding andactivating VEGFR1, but do block VEGF from binding and activating VEGFR2.The anti-tumor effects of such VEGFR2 inhibition are clearlydemonstrated. These results show VEGFR2 to be the VEGF receptor thatmediates permeability and highlight its role in tumor angiogenesis.

This invention therefore further validates VEGF inhibition as therapyfor the treatment of solid tumors. More importantly, the inventionprovides a range of new VEGFR2-blocking, human anti-VEGF antibodies fortherapeutic intervention and, in particular, for use as safe andeffective drugs for inhibiting angiogenesis in tumors and otherdiseases.

The benefits of the present invention are not limited to the lack ofside effects. Although these are important features that will havenotable benefits, particularly in the treatment of children and patientswith bone disorders, the antibodies of the invention have numerous otheradvantages.

For example, the VEGFR2-blocking, human anti-VEGF antibodies of thepresent invention have important advantages in inhibiting thedetrimental actions of tumor-associated macrophages. It is now knownthat tumor-associated macrophages play important roles in cancer, bothin the initial development stages and in tumor progression andmetastasis. As detailed below, the human antibodies of this inventionare ideally suited to counteracting the adverse effects of thesemacrophages.

The formation of a tumor vasculature and/or access to the hostvasculature is a crucial step in the development of malignant tumors.Indeed, the formation of a high-density vessel network, termed “theangiogenic switch”, is closely associated with the transition tomalignancy (Hanahan and Folkman, 1996). It is now known that macrophagesassociated with the primary tumor play a key role in both the angiogenicswitch and the progression to malignancy (Lin et al., 2006).Furthermore, it has been shown that inhibiting macrophage infiltrationinto tumors delays the angiogenic switch and malignant transition (Linet al., 2006).

In many patients with cancer, metastasis is the ultimate cause of death.Invasion of tumor cells from the primary tumor into the surroundingconnective tissue and blood vessels is a key step in the metastaticprocess. Macrophages were earlier reported to be associated with tumorprogression and metastasis (Lin et al., 2001). Subsequent studies haveshown that the interaction between tumor cells and macrophagesfacilitates the migration of carcinoma cells in the primary tumor, andthat this process involves a paracrine loop (Wyckoff et al., 2004;Goswami et al., 2005).

Moreover, tumor-infiltrating or tumor-associated macrophages are nowknown to be prominent in various tumor microenvironments, includingareas of invasion, stromal and perivascular areas and avascular andperinecrotic areas. The actions of macrophages in each of these tumormicroenvironments stimulate tumor progression and metastasis bypromoting cancer cell motility, metastasis and angiogenesis,respectively (Lewis and Pollard, 2006). Therefore, macrophages haverecently become an important target in the battle against cancer(Condeelis and Pollard, 2006).

In this regard, the human antibodies of the present invention haveimportant advantages as they block activation of VEGFR2 and thus reducemacrophage infiltration into tumors, and can therefore reduce thetransition to malignancy, tumor progression and/or metastasis. This isvalidated by results from animal studies presented herein showing thattumor-associated macrophages express VEGFR2, and that VEGFR2 mediatesthe VEGF-induced chemotaxis of these cells. It is also shown herein thatthe selective blockade of VEGFR2 caused by the human antibodies of thisinvention exerts a potent anti-cancer effect. This anti-cancer effect isaccompanied by a reduction in macrophage infiltration into the tumor,indicating that selectively blocking the VEGF-VEGFR2 interaction in hostmacrophages contributes to the observed therapeutic effects.

The VEGFR2-blocking, human anti-VEGF antibodies of this invention alsohave advantages in connection with reducing lymphatic vessel density intumors. In addition to egress of tumor cells into tumor blood vessels,metastasis is facilitated by lymphangiogenesis, i.e., the growth of newintratumoral or peritumoral lymphatic vessels from pre-existing vessels.Indeed, in several types of cancer, including breast cancer, escape oftumor cells via the lymphatic system is believed to be the predominantmeans by which malignant cells from the primary tumor are seeded todistant sites.

For several years, it was thought that lymphangiogenesis was primarilyinduced by VEGF-C and/or VEGF-D. However, a body of evidence has nowaccumulated implicating VEGF-A in lymphangiogenesis. Moreover, recentstudies have shown that murine antibodies against VEGF-A are effectivein inhibiting tumor lymphangiogenesis and metastases in vivo (Whitehurstet al., 2007).

Results are presented herein to show that the VEGFR2-blocking, humananti-VEGF antibodies of this invention do, indeed, reduce tumorlymphatic vessel density (FIG. 18A, FIG. 18B and FIG. 18C). The humanantibodies of the present invention will therefore inhibit tumorlymphangiogenesis and provide the additional benefit of reducingmetastases via the lymphatic route, as well as inhibiting angiogenesisand metastatic escape via tumor blood vessels. It can be seen,therefore, that the human antibodies of the present invention have theability to reduce metastasis or metastatic events via several points ofintervention.

Moreover, the data in FIG. 18A show that the antibodies of the presentinvention reduce tumor lymphatic vessel density as measured by areduction in podoplanin and in PROX1. As podoplanin is a marker for softtissue cancers, such as chondrosarcoma, and for lymphatic tumors, suchas follicular dendritic cell sarcoma) (Xie et al., 2008), and as PROX1has been implicated in predicting the invasiveness of colon cancer(Petrova et al., 2008), this emphasizes the use of the VEGFR2-blocking,human anti-VEGF antibodies of the invention to treat those particulardisease indications.

A further advantageous property shown by the VEGFR2-blocking, humananti-VEGF antibodies of this invention is the ability to significantlyreduce infiltration or recruitment of myeloid-derived suppressor cells,in particular CD11b+/Gr1+ cells, into tumors. Furthermore, this propertyis not shown by the 2C3 antibody and only at a more reduced level byAvastin. Preferred antibodies of the invention can decrease theinfiltration or recruitment of CD11b+/Gr1+ cells into tumors (e.g.decrease the number of double-positive cells present in tumors) by 30%or more, preferably by 32%, 34%, 36%, 38% or more, compared to a controllevel (e.g. an untreated tumor or a tumor treated with a controlantibody).

Thus, further studies in MDA-MB-231 tumor-bearing mice have shown thatsignificantly less CD11b/Gr1 double positive cells infiltrate tumors inr84-treated animals as opposed to control. In comparative studies,neither the 2C3 antibody nor Avastin showed a statistically significantdecrease in CD11b+/Gr1+ infiltration, although some reduction wasmeasurable in Avastin-treated animals. The reduction in the number ofdouble positive cells observed is 39% (FIG. 25).

The reduced infiltration of myeloid derived suppressor cells CD11b+/Gr1+is of special interest, as cells expressing both markers have recentlybeen associated with mediation of tumor refractoriness to anti-VEGFtherapy (Shojaei et al., 2007). Myeloid-derived suppressor cells(CD11b+Gr1+) are also an important contributor to tumor progression. Inthe tumor microenvironment these cells secrete immunosuppressivemediators and induce T-lymphocyte dysfunction (Gabrilovich et al., 2001;Serafini et al., 2004).

As CD11b+/Gr1+ cells are associated with tumor refractoriness toanti-VEGF therapy and contribute to tumor progression, the effect of theantibodies of the invention to reduce infiltration of these cells intotumors clearly has a potential importance for therapeutic applicationsof the antibodies of the invention, in particular therapeuticapplications related to the treatment of angiogenic diseases, includingcancer.

Indeed, as the results herein show that the tumor infiltration ofCD11b+/Gr1+ cells is least pronounced/significantly lower in the animalstreated with antibodies of the invention, it suggests that treatmentwith antibodies of the invention is likely to be less prone to thedevelopment of drug resistance or refractoriness to anti-VEGF therapythan treatment with other drugs targeting VEGF, e.g. other anti-VEGFantibodies. In addition, given the proposed role of CD11b+/Gr1+ cells intumor progression, the ability of the antibodies of the invention toreduce infiltration or recruitment of such cells into tumors might wellform part of the mechanism involved in the anti-tumor activity, e.g. theinhibition of tumor growth shown by the antibodies of the invention.

The VEGFR2-blocking, human anti-VEGF antibodies of this invention havealso been shown to not induce toxicity when administered chronically inin vivo mouse models.

The VEGFR2-blocking, human anti-VEGF antibodies of this inventionpreferably have the advantageous property of binding to mouse VEGF andhuman VEGF. The ability to bind mouse VEGF is an important advantageover the antibodies 2C3 and Avastin.

In addition, antibody conjugates based upon the VEGFR2-blocking, humananti-VEGF antibodies of the present invention can be used to delivertherapeutic agents to the tumor environment, whereas many otheranti-VEGF antibodies cannot. The human antibodies of the invention bindto both tumor vasculature and tumor stroma upon administration in vivo,but do not bind to vasculature or connective tissue in normal organs ortissues. Therapeutic constructs based upon the present human antibodiestherefore have the advantage of combining two functions within onemolecule: the anti-angiogenic properties of the antibody or fragmentthereof and the properties of the therapeutic agent selected forattachment. In summary, a human antibody of the present invention may beused both as an anti-angiogenic agent and a vascular targeting agent,whereas many anti-VEGF antibodies of the prior art cannot be used in avascular targeting capacity.

As VEGFR2 is the key receptor on endothelium, blocking VEGF binding toVEGFR2 is critical for an anti-angiogenic effect. Although VEGFR1 isexpressed on endothelium, it is non-signal transducing, or passive, inthis context. Therefore, the inability of the human antibodies of thepresent invention to block VEGF binding to VEGFR1 is without consequenceto their effectiveness as anti-angiogenic and anti-tumor agents. Infact, rather than inhibiting VEGF binding to VEGFR1, which occurs withthe blocking antibodies of the prior art, the ability of the presenthuman antibodies to bind to VEGF and yet to not substantially disturbVEGF-VEGFR1 interactions enhances the drug delivery properties of thesenew antibodies.

The present inventors realized that blocking antibodies would still beexpected to function to deliver therapeutic agents to the tumorenvironment by binding to tumor-localized VEGF that is not bound to areceptor. Specifically, they understood that such human antibodies willbind to VEGF in the tumor stroma and deliver therapeutic agents thereto.This provides a reservoir of drug around the endothelium, causingcytotoxic or other destructive effects on the vascular endothelial cellsand exerting an anti-tumor effect.

The VEGF associated with the stroma or connective tissue is not bound toa VEGF receptor in a classic sense, i.e., a cell surface receptor.Rather, VEGF is bound to one or more connective tissue components,including proteoglycans, such as heparan sulfate proteoglycan, through abasic region of VEGF. These sequences (and the exons encoding them) aremissing in VEGF121 protein (and underlying DNA), so this isoform shouldnot be present in stroma in significant amounts. VEGF in the tumorstroma is often termed “free”, although it is still localized within thetumor, so “free” essentially means non-receptor-bound.

The inventors further deduced that a human antibody that blocks VEGFbinding to one, but not both receptors, would still be able to delivertherapeutic agents to the tumor environment by binding to receptor boundVEGF on the vasculature. This is one of the advantageous features of thepresent invention. Namely, the provision of human antibodies that blockVEGF binding to VEGFR2, and hence inhibit the angiogenic signal fromVEGF, but that do not block VEGF binding to VEGFR1. In addition toreducing systemic side effects by maintaining VEGF signaling via VEGFR1in other cell types and tissues, these human antibodies are able tolocalize to VEGF-VEGFR1 complex on tumor vasculature and to delivertherapeutic agents directly thereto.

Both VEGFR1 and VEGFR2 are upregulated on tumor endothelial cells, asopposed to endothelial cells in normal tissues. VEGFR1 is highlyexpressed on tumor vascular endothelium, which makes the targetingaspects of the present invention particularly effective. In fact,VEGFR1, although “non-signaling” in endothelium, is expressed at leastat the same levels as VEGFR2, if not at higher levels. A factorunderlying this phenomenon is that VEGFR1 is upregulated in response toboth hypoxia and VEGF, whereas VEGFR2 is only upregulated in response toVEGF and is not influenced by hypoxia.

Although the role of VEGFR1 on endothelium remains uncertain, VEGFR1 mayact as a decoy receptor to “capture” VEGF and pass the ligand onto thesignaling receptor, VEGFR2. For this to be true, one would expect thedecoy receptor to have a higher affinity for VEGF than the signalingreceptor, which is indeed the case. In light of this, and perhaps alsodue to enhanced expression levels, the VEGFR2-blocking,non-VEGFR1-blocking human antibodies of this invention are idealdelivery agents for tumor treatment. Therapeutic conjugates of theseantibodies are able to simultaneously inhibit angiogenesis throughVEGFR2 and destroy the existing vasculature by delivering a therapeuticagent to VEGF-VEGFR1 receptor complex.

The inventors are by no means limited to the foregoing scientificreasoning as an explanation for the beneficial anti-angiogenic andtumor-localizing properties of the present human antibodies. Althoughthe utility of the invention is self-evident and needs no underlyingtheory to be put into practice, the inventors have consideredalternative mechanisms by which VEGFR2-blocking, non-VEGFR1-blockinghuman antibodies may effectively and specifically localize to tumorvasculature.

Such human antibodies could bind to VEGF that is associated with anotherknown or, as yet, uncharacterized VEGF binding protein on the cellsurface or could bind VEGF that is bound to heparan sulfateproteoglycans on the surface of endothelial cells. Antibody localizationcould also be enhanced by binding to another member of the VEGF familyof proteins, i.e., VEGF-B, VEGF-C, VEGF-D, which are associated with theblood vessels, although this is less likely.

Another advantageous property of the VEGFR2-blocking, human anti-VEGFantibodies of the invention is that these antibodies neutralize thesurvival signal or “protective effect” of VEGF, which is mediatedthrough VEGFR2. In addition to making the human antibodies moreeffective themselves, this property makes them particularly useful incombination with other agents that are hampered by VEGF's survivalfunction.

For example, VEGF protects the endothelium from radiotherapy. Therefore,both the naked antibodies and immunoconjugates of the present inventionare ideal for use in combination with radiotherapy. Even more benefitsare provided by the use of such a human antibody attached to aradiotherapeutic agent. This type of construct would have the tripleadvantages of: (1) exerting an anti-angiogenic effect through theantibody portion; (2) exerting a tumor vasculature destructive effectthrough delivery of the radiotherapeutic agent; and (3) preventingVEGF's typical survival signal from counteracting the effects of theradiotherapeutic agent.

Other constructs with similarly synergistic effects are VEGFR2-blocking,human anti-VEGF antibodies in association with anti-tubulin drugs orprodrugs, anti-apoptopic agents and other anti-angiogenic agents. Theactions of agents or drugs that cause apoptosis are antagonized by VEGF.The present invention therefore improves the effectiveness of suchagents by neutralizing VEGF. VEGF survival signals also opposeendostatin, limiting this therapy. Therefore, in combined use withendostatin, the VEGFR2-blocking, human anti-VEGF antibodies of theinvention will neutralize VEGF and amplify the anti-tumor effects ofendostatin. The VEGFR2-blocking, human anti-VEGF antibodies may also beused to specifically delivery collagenase to the tumor, where thecollagenase will produce endostatin in situ, achieving similar benefits.

In all such enhanced or synergistic combinations, the human antibodiesand other agents may be administered separately, or the second agentsmay be linked to the human antibodies for specific delivery (i.e.,targeted delivery to VEGFR1). In combinations with endostatin, chemicalconjugates or recombinant fusion proteins will be preferred, as thesewill counteract the short half life of endostatin, which is currently alimitation of potential endostatin therapy. Combinations with, ortargeted forms of, tissue plasminogen activator (tPA) may also beemployed.

Further advantages of the human therapeutics of the present inventioninclude the ability to lower the interstitial pressure. As VEGF-mediatedincreased permeability contributes to the interstitial pressure, reducedsignaling via VEGFR2 will reduce both permeability and interstitialpressure. This, in turn, will reduce the barrier to drugs traversing theentirety of the tumor tissue, so that tumor cells distant from thevasculature can be killed. Prolonged therapy can also be achieved as thepresent compositions with have no, negligible or low immunogenicity.

B3. Antibody CDR Sequences

The term “variable”, as used herein in reference to antibodies, meansthat certain portions of the variable domains differ extensively insequence among antibodies, and are used in the binding and specificityof each particular antibody to its particular antigen. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments termed “hypervariableregions”, both in the light chain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework region (FR). The variable domains of native heavy and lightchains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively),largely adopting a β-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some cases,forming part of, the β-sheet structure.

The hypervariable regions in each chain are held together in closeproximity by the FRs and, with the hypervariable regions from the otherchain, contribute to the formation of the antigen-binding site ofantibodies (Kabat et al., 1991, specifically incorporated herein byreference). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

The term “hypervariable region”, as used herein, refers to the aminoacid residues of an antibody that are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-56 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., 1991, specifically incorporated herein by reference)and/or those residues from a “hypervariable loop” (i.e. residues 26-32(L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain). “Framework” or “FR” residues are those variable domain residuesother than the hypervariable region residues as herein defined.

The DNA and deduced amino acid sequences of the VH and VL chains of ther84 ScFv fragment are provided herein as SEQ ID NO:1 (VH, nucleic acid),SEQ ID NO:2 (VL, nucleic acid) SEQ ID NO:3 (VH, amino acid) and SEQ IDNO:4 (VL, amino acid). The DNA sequences of the VH and VL chains of ther84 full length IgG are provided herein as SEQ ID NO:26 (VH, nucleicacid) and SEQ ID NO:27 (VL, nucleic acid). These sequences encompassCDR1-3 of the variable regions of the heavy and light chains of theantibody.

As described herein (Section C7), with the provision of structural andfunctional information for a biological molecule, a range of equivalent,or even improved molecules can be generated. This applies to theVEGFR2-blocking, human anti-VEGF antibodies of the present invention, asexemplified by the r84 antibody. Although antigen-binding and otherfunctional properties of an antibody must be conserved, there is anextremely high degree of skill in the art in making equivalent and evenimproved antibodies once a reference antibody has been provided. Suchtechnical skill can, in light of the sequences and information providedherein, be applied to the production of further antibodies that havelike, improved or otherwise desirable characteristics.

For equivalent antibodies, certain amino acids may substituted for otheramino acids in the antibody constant or variable domain frameworkregions without appreciable loss of interactive binding capacity. It ispreferable that such changes be made in the DNA sequences encoding theantibody portions and that the changes be conservative in nature (seeSection C7, the codon information in Table A, and the supportingtechnical details on site-specific mutagenesis). Naturally, there is alimit to the number of changes that should be made, but this will beknown those of ordinary skill in the art.

Other types of variants are antibodies with improved biologicalproperties relative to the parent antibody from which they aregenerated. Such variants, or second generation compounds, are typicallysubstitutional variants involving one or more substituted hypervariableregion residues of a parent antibody. A convenient way for generatingsuch substitutional variants is affinity maturation using phage display.

In affinity maturation using phage display, several hypervariable regionsites (e.g. 6-7 sites) are mutated to generate all possible aminosubstitutions at each site. The antibody variants thus generated aredisplayed in a monovalent fashion from filamentous phage particles asfusions to the gene III product of M13 packaged within each particle.The phage-displayed variants are then screened for their biologicalactivity (e.g. binding affinity) as herein disclosed. In order toidentify candidate hypervariable region sites for modification, alaninescanning mutagenesis can be performed to identify hypervariable regionresidues contributing significantly to antigen binding.

Alternatively, or in addition, it is contemplated that the crystalstructure of the antigen-antibody complex be delineated and analyzed toidentify contact points between the antibody and VEGF. Such contactresidues and neighboring residues are candidates for substitution. Oncesuch variants are generated, the panel of variants is subjected toscreening, as described herein, and antibodies with analogues butdifferent or even superior properties in one or more relevant assays areselected for further development.

Further aspects of the invention therefore concern isolated or purifiedDNA segments and recombinant vectors encoding CDR regions ofVEGFR2-blocking, human anti-VEGF antibody heavy and light chains of theinvention, such as r84 heavy and light chains, and the creation and useof recombinant host cells through the application of DNA technology,that express such CDR regions.

The present invention thus concerns human or synthetic DNA segments,which are free from total genomic DNA and are capable of expressing CDRregions of VEGFR2-blocking, human anti-VEGF antibody heavy and/or lightchains of the invention, such as r84 heavy and/or light chains. As usedherein, the term “DNA segment” refers to a DNA molecule that has beenisolated or purified free of total genomic DNA of a particular species.Included within the term “DNA segment”, are DNA segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phage, viruses, and the like.

Similarly, a DNA segment comprising a coding segment or isolated orpurified gene portion encoding purified CDR regions of VEGFR2-blocking,human anti-VEGF antibody heavy and/or light chains of the invention,such as r84 heavy and/or light chains, refers to a DNA segment includingsuch coding sequences and, in certain aspects, regulatory sequences,isolated or purified substantially away from other naturally occurringgenes or protein encoding sequences. In this respect, the term “gene” isused for simplicity to refer to a functional protein, polypeptide orpeptide encoding unit. As will be understood by those in the art, thisfunctional term includes the native antibody-encoding sequences andsmaller engineered segments that express, or may be adapted to express,suitable antigen binding proteins, polypeptides or peptides.

“Isolated or purified substantially away from other coding sequences”means that the coding segment or isolated gene portion of interest formsthe significant part of the coding region of the DNA segment, and thatthe DNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or cDNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated or purifiedcoding segments or isolated or purified gene portions and recombinantvectors incorporating DNA sequences that encode CDR regions ofVEGFR2-blocking, human anti-VEGF antibody heavy and/or light chains ofthe invention, such as r84 heavy and/or light chains, that comprise atleast a first sequence region that includes an amino acid sequenceregion of at least about 75%, more preferably, at least about 80%, morepreferably, at least about 85%, more preferably, at least about 90% andmost preferably, at least about 95% or so amino acid sequence identityto the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4; wherein saidCDR regions at least substantially maintain the biological properties ofthe CDR regions of amino acid sequences SEQ ID NO:3 or SEQ ID NO:4.

As disclosed herein, the sequences may comprise certain biologicallyfunctional equivalent amino acids or “conservative substitutions”. Othersequences may comprise functionally non-equivalent amino acids or“non-conservative substitutions” deliberately engineered to improve theproperties of the CDR or antibody containing the CDR, as is known thoseof ordinary skill in the art and further described herein.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still correspond to asequence of the invention, so long as the sequence meets the criteriaset forth above, preferably including the maintenance or improvement ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences includes various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region, and alsocontrol regions.

The nucleic acid segments of the present invention may thus be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol.

Recombinant vectors therefore form further aspects of the presentinvention. Particularly useful vectors are contemplated to be thosevectors in which the coding portion of the DNA segment is positionedunder the control of a promoter. Generally, although not exclusively, arecombinant or heterologous promoter will be employed, i.e., a promoternot normally associated with coding sequences in their naturalenvironment. Such promoters may include bacterial, viral, eukaryotic andmammalian promoters, so long as the promoter effectively directs theexpression of the DNA segment in the cell type, organism, or evenanimal, chosen for expression.

The use of promoter and cell type combinations for protein expression isknown to those of skill in the art of molecular biology. The promotersemployed may be constitutive, or inducible, and can be used under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides.

The expression of the nucleic acid sequences of the invention may beconveniently achieved by any one or more standard techniques known thoseof ordinary skill in the art and further described herein. For example,the later description of the recombinant expression of fusion proteinsapplies equally well to antibodies and antibody fragments that are notoperatively associated with another coding sequence at the nucleic acidlevel.

B4. Antibodies from Phagemid Libraries

Recombinant technology now allows the preparation of antibodies havingthe desired specificity from recombinant genes encoding a range ofantibodies (Van Dijk et al., 1989; incorporated herein by reference).Certain recombinant techniques involve the isolation of the antibodygenes by immunological screening of combinatorial immunoglobulin phageexpression libraries prepared from RNA isolated from the spleen of animmunized animal (Morrison et al., 1986; Winter and Milstein, 1991; eachincorporated herein by reference).

For such methods, combinatorial immunoglobulin phagemid libraries areprepared from RNA isolated from the spleen of the immunized animal, andphagemids expressing appropriate antibodies are selected by panningusing cells expressing the antigen and control cells. The advantages ofthis approach over conventional hybridoma techniques are thatapproximately 10⁴ times as many antibodies can be produced and screenedin a single round, and that new specificities are generated by H and Lchain combination, which further increases the percentage of appropriateantibodies generated.

One method for the generation of a large repertoire of diverse antibodymolecules in bacteria utilizes the bacteriophage lambda as the vector(Huse et al., 1989; incorporated herein by reference). Production ofantibodies using the lambda vector involves the cloning of heavy andlight chain populations of DNA sequences into separate starting vectors.The vectors are subsequently combined randomly to form a single vectorthat directs the co-expression of heavy and light chains to formantibody fragments. The heavy and light chain DNA sequences are obtainedby amplification, preferably by PCR™ or a related amplificationtechnique, of mRNA isolated from spleen cells (or hybridomas thereof)from an animal that has been immunized with a selected antigen. Theheavy and light chain sequences are typically amplified using primersthat incorporate restriction sites into the ends of the amplified DNAsegment to facilitate cloning of the heavy and light chain segments intothe starting vectors.

Another method for the generation and screening of large libraries ofwholly or partially synthetic antibody combining sites, or paratopes,utilizes display vectors derived from filamentous phage such as M13, flor fd. These filamentous phage display vectors, referred to as“phagemids”, yield large libraries of monoclonal antibodies havingdiverse and novel immunospecificities. The technology uses a filamentousphage coat protein membrane anchor domain as a means for linkinggene-product and gene during the assembly stage of filamentous phagereplication, and has been used for the cloning and expression ofantibodies from combinatorial libraries (Kang et al., 1991; Barbas etal., 1991; each incorporated herein by reference).

This general technique for filamentous phage display is described inU.S. Pat. No. 5,658,727, incorporated herein by reference. In a mostgeneral sense, the method provides a system for the simultaneous cloningand screening of pre-selected ligand-binding specificities from antibodygene repertoires using a single vector system. Screening of isolatedmembers of the library for a pre-selected ligand-binding capacity allowsthe correlation of the binding capacity of an expressed antibodymolecule with a convenient means to isolate the gene that encodes themember from the library.

Linkage of expression and screening is accomplished by the combinationof targeting of a fusion polypeptide into the periplasm of a bacterialcell to allow assembly of a functional antibody, and the targeting of afusion polypeptide onto the coat of a filamentous phage particle duringphage assembly to allow for convenient screening of the library memberof interest. Periplasmic targeting is provided by the presence of asecretion signal domain in a fusion polypeptide. Targeting to a phageparticle is provided by the presence of a filamentous phage coat proteinmembrane anchor domain (i.e., a cpIII- or cpVIII-derived membrane anchordomain) in a fusion polypeptide.

The diversity of a filamentous phage-based combinatorial antibodylibrary can be increased by shuffling of the heavy and light chaingenes, by altering one or more of the complementarity determiningregions of the cloned heavy chain genes of the library, or byintroducing random mutations into the library by error-prone polymerasechain reactions. Additional methods for screening phagemid libraries aredescribed in U.S. Pat. Nos. 5,580,717; 5,427,908; 5,403,484; and5,223,409, each incorporated herein by reference.

Another method for the screening of large combinatorial antibodylibraries has been developed, utilizing expression of populations ofdiverse heavy and light chain sequences on the surface of a filamentousbacteriophage, such as M13, fl or fd (U.S. Pat. No. 5,698,426;incorporated herein by reference). Two populations of diverse heavy (Hc)and light (Lc) chain sequences are synthesized by polymerase chainreaction (PCR™). These populations are cloned into separate M13-basedvector containing elements necessary for expression. The heavy chainvector contains a gene VIII (gVIII) coat protein sequence so thattranslation of the heavy chain sequences produces gVIII-Hc fusionproteins. The populations of two vectors are randomly combined such thatonly the vector portions containing the Hc and Lc sequences are joinedinto a single circular vector.

The combined vector directs the co-expression of both Hc and Lcsequences for assembly of the two polypeptides and surface expression onM13 (U.S. Pat. No. 5,698,426; incorporated herein by reference). Thecombining step randomly brings together different Hc and Lc encodingsequences within two diverse populations into a single vector. Thevector sequences donated from each independent vector are necessary forproduction of viable phage. In addition, since the pseudo gVIIIsequences are contained in only one of the two starting vectors,co-expression of functional antibody fragments as Lc associated gVIII-Hcfusion proteins cannot be accomplished on the phage surface until thevector sequences are linked in the single vector.

Surface expression of the antibody library is performed in an ambersuppressor strain. An amber stop codon between the Hc sequence and thegVIII sequence unlinks the two components in a non-suppressor strain.Isolating the phage produced from the non-suppressor strain andinfecting a suppressor strain will link the Hc sequences to the gVIIIsequence during expression. Culturing the suppressor strain afterinfection allows the coexpression on the surface of M13 of all antibodyspecies within the library as gVIII fusion proteins (gVIII-Fab fusionproteins). Alternatively, the DNA can be isolated from thenon-suppressor strain and then introduced into a suppressor strain toaccomplish the same effect.

The surface expression library is screened for specific Fab fragmentsthat bind preselected molecules by standard affinity isolationprocedures. Such methods include, for example, panning (Parmley andSmith, 1988; incorporated herein by reference), affinity chromatographyand solid phase blotting procedures. Panning is preferred, because hightiters of phage can be screened easily, quickly and in small volumes.Furthermore, this procedure can select minor Fab fragments specieswithin the population, which otherwise would have been undetectable, andamplified to substantially homogenous populations. The selected Fabfragments can be characterized by sequencing the nucleic acids encodingthe polypeptides after amplification of the phage population.

Another method for producing diverse libraries of antibodies andscreening for desirable binding specificities is described in U.S. Pat.Nos. 5,667,988 and 5,759,817, each incorporated herein by reference. Themethod involves the preparation of libraries of heterodimericimmunoglobulin molecules in the form of phagemid libraries usingdegenerate oligonucleotides and primer extension reactions toincorporate the degeneracies into the CDR regions of the immunoglobulinvariable heavy and light chain variable domains, and display of themutagenized polypeptides on the surface of the phagemid. Thereafter, thedisplay protein is screened for the ability to bind to a preselectedantigen.

The method for producing a heterodimeric immunoglobulin moleculegenerally involves (1) introducing a heavy or light chain Vregion-coding gene of interest into the phagemid display vector; (2)introducing a randomized binding site into the phagemid display proteinvector by primer extension with an oligonucleotide containing regions ofhomology to a CDR of the antibody V region gene and containing regionsof degeneracy for producing randomized coding sequences to form a largepopulation of display vectors each capable of expressing differentputative binding sites displayed on a phagemid surface display protein;(3) expressing the display protein and binding site on the surface of afilamentous phage particle; and (4) isolating (screening) thesurface-expressed phage particle using affinity techniques such aspanning of phage particles against a preselected antigen, therebyisolating one or more species of phagemid containing a display proteincontaining a binding site that binds a preselected antigen.

A further variation of this method for producing diverse libraries ofantibodies and screening for desirable binding specificities isdescribed in U.S. Pat. No. 5,702,892, incorporated herein by reference.In this method, only heavy chain sequences are employed, the heavy chainsequences are randomized at all nucleotide positions that encode eitherthe CDR1 or CDRIII hypervariable region, and the genetic variability inthe CDRs is generated independent of any biological process.

In the method, two libraries are engineered to genetically shuffleoligonucleotide motifs within the framework of the heavy chain genestructure. Through random mutation of either CDRI or CDRIII, thehypervariable regions of the heavy chain gene were reconstructed toresult in a collection of highly diverse sequences. The heavy chainproteins encoded by the collection of mutated gene sequences possessedthe potential to have all of the binding characteristics of animmunoglobulin while requiring only one of the two immunoglobulinchains.

Specifically, the method is practiced in the absence of theimmunoglobulin light chain protein. A library of phage displayingmodified heavy chain proteins is incubated with an immobilized ligand toselect clones encoding recombinant proteins that specifically bind theimmobilized ligand. The bound phage are then dissociated from theimmobilized ligand and amplified by growth in bacterial host cells.Individual viral plaques, each expressing a different recombinantprotein, are expanded, and individual clones can then be assayed forbinding activity.

B5. Transgenic Mice Containing Human Antibody Libraries

Recombinant technology is now available for the preparation ofantibodies. In addition to the combinatorial immunoglobulin phageexpression libraries disclosed above, another molecular cloning approachis to prepare antibodies from transgenic mice containing human antibodylibraries. Such techniques are described in U.S. Pat. No. 5,545,807,incorporated herein by reference.

In a most general sense, these methods involve the production of atransgenic animal that has inserted into its germline genetic materialthat encodes for at least part of an immunoglobulin of human origin orthat can rearrange to encode a repertoire of immunoglobulins. Theinserted genetic material may be produced from a human source, or may beproduced synthetically. The material may code for at least part of aknown immunoglobulin or may be modified to code for at least part of analtered immunoglobulin.

The inserted genetic material is expressed in the transgenic animal,resulting in production of an immunoglobulin derived at least in partfrom the inserted human immunoglobulin genetic material. It is found thegenetic material is rearranged in the transgenic animal, so that arepertoire of immunoglobulins with part or parts derived from insertedgenetic material may be produced, even if the inserted genetic materialis incorporated in the germline in the wrong position or with the wronggeometry.

The inserted genetic material may be in the form of DNA cloned intoprokaryotic vectors such as plasmids and/or cosmids. Larger DNAfragments are inserted using yeast artificial chromosome vectors (Burkeet al., 1987; incorporated herein by reference), or by introduction ofchromosome fragments (Richer and Lo, 1989; incorporated herein byreference). The inserted genetic material may be introduced to the hostin conventional manner, for example by injection or other proceduresinto fertilized eggs or embryonic stem cells.

In preferred aspects, a host animal that initially does not carrygenetic material encoding immunoglobulin constant regions is utilized,so that the resulting transgenic animal will use only the inserted humangenetic material when producing immunoglobulins. This can be achievedeither by using a naturally occurring mutant host lacking the relevantgenetic material, or by artificially making mutants e.g., in cell linesultimately to create a host from which the relevant genetic material hasbeen removed.

Where the host animal carries genetic material encoding immunoglobulinconstant regions, the transgenic animal will carry the naturallyoccurring genetic material and the inserted genetic material and willproduce immunoglobulins derived from the naturally occurring geneticmaterial, the inserted genetic material, and mixtures of both types ofgenetic material. In this case the desired immunoglobulin can beobtained by screening hybridomas derived from the transgenic animal,e.g., by exploiting the phenomenon of allelic exclusion of antibody geneexpression or differential chromosome loss.

Once a suitable transgenic animal has been prepared, the animal issimply immunized with the desired immunogen. Depending on the nature ofthe inserted material, the animal may produce a chimeric immunoglobulin,e.g. of mixed mouse/human origin, where the genetic material of foreignorigin encodes only part of the immunoglobulin; or the animal mayproduce an entirely foreign immunoglobulin, e.g. of wholly human origin,where the genetic material of foreign origin encodes an entireimmunoglobulin.

Polyclonal antisera may be produced from the transgenic animal followingimmunization Immunoglobulin-producing cells may be removed from theanimal to produce the immunoglobulin of interest. Preferably, monoclonalantibodies are produced from the transgenic animal, e.g., by fusingspleen cells from the animal with myeloma cells and screening theresulting hybridomas to select those producing the desired antibody.Suitable techniques for such processes are described herein.

In an alternative approach, the genetic material may be incorporated inthe animal in such a way that the desired antibody is produced in bodyfluids such as serum or external secretions of the animal, such as milk,colostrum or saliva. For example, by inserting in vitro genetic materialencoding for at least part of a human immunoglobulin into a gene of amammal coding for a milk protein and then introducing the gene to afertilized egg of the mammal, e.g., by injection, the egg may developinto an adult female mammal producing milk containing immunoglobulinderived at least in part from the inserted human immunoglobulin geneticmaterial. The desired antibody can then be harvested from the milk.Suitable techniques for carrying out such processes are known to thoseskilled in the art.

The foregoing transgenic animals are usually employed to produce humanantibodies of a single isotype, more specifically an isotype that isessential for B cell maturation, such as IgM and possibly IgD. Anotherpreferred method for producing human anti-VEGF antibodies is to use thetechnology described in U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016; and 5,770,429; each incorporated by reference,wherein transgenic animals are described that are capable of switchingfrom an isotype needed for B cell development to other isotypes.

In the development of a B lymphocyte, the cell initially produces IgMwith a binding specificity determined by the productively rearrangedV_(H) and V_(L) regions. Subsequently, each B cell and its progeny cellssynthesize antibodies with the same L and H chain V regions, but theymay switch the isotype of the H chain. The use of mu or delta constantregions is largely determined by alternate splicing, permitting IgM andIgD to be coexpressed in a single cell. The other heavy chain isotypes(gamma, alpha, and epsilon) are only expressed natively after a generearrangement event deletes the C mu and C delta exons. This generearrangement process, termed isotype switching, typically occurs byrecombination between so called switch segments located immediatelyupstream of each heavy chain gene (except delta). The individual switchsegments are between 2 and 10 kb in length, and consist primarily ofshort repeated sequences.

For these reasons, it is preferable that transgenes incorporatetranscriptional regulatory sequences within about 1-2 kb upstream ofeach switch region that is to be utilized for isotype switching. Thesetranscriptional regulatory sequences preferably include a promoter andan enhancer element, and more preferably include the 5′ flanking (i.e.,upstream) region that is naturally associated (i.e., occurs in germlineconfiguration) with a switch region. Although a 5′ flanking sequencefrom one switch region can be operably linked to a different switchregion for transgene construction, in some embodiments it is preferredthat each switch region incorporated in the transgene construct have the5′ flanking region that occurs immediately upstream in the naturallyoccurring germline configuration. Sequence information relating toimmunoglobulin switch region sequences is known (Mills et al., 1990;Sideras et al., 1989; each incorporated herein by reference).

In the method described in U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,661,016; and 5,770,429, the human immunoglobulintransgenes contained within the transgenic animal function correctlythroughout the pathway of B-cell development, leading to isotypeswitching. Accordingly, in this method, these transgenes are constructedso as to produce isotype switching and one or more of the following: (1)high level and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

An important requirement for transgene function is the generation of aprimary antibody repertoire that is diverse enough to trigger asecondary immune response for a wide range of antigens. The rearrangedheavy chain gene consists of a signal peptide exon, a variable regionexon and a tandem array of multi-domain constant region regions, each ofwhich is encoded by several exons. Each of the constant region genesencode the constant portion of a different class of immunoglobulins.During B-cell development, V region proximal constant regions aredeleted leading to the expression of new heavy chain classes. For eachheavy chain class, alternative patterns of RNA splicing give rise toboth transmembrane and secreted immunoglobulins.

The human heavy chain locus consists of approximately 200 V genesegments spanning 2 Mb, approximately 30 D gene segments spanning about40 kb, six J segments clustered within a 3 kb span, and nine constantregion gene segments spread out over approximately 300 kb. The entirelocus spans approximately 2.5 Mb of the distal portion of the long armof chromosome 14. Heavy chain transgene fragments containing members ofall six of the known V_(H) families, the D and J gene segments, as wellas the mu, delta, gamma 3, gamma 1 and alpha 1 constant regions areknown (Berman et al., 1988; incorporated herein by reference). Genomicfragments containing all of the necessary gene segments and regulatorysequences from a human light chain locus is similarly constructed.

The expression of successfully rearranged immunoglobulin heavy and lighttransgenes usually has a dominant effect by suppressing therearrangement of the endogenous immunoglobulin genes in the transgenicnonhuman animal. However, in certain embodiments, it is desirable toeffect complete inactivation of the endogenous Ig loci so that hybridimmunoglobulin chains comprising a human variable region and a non-human(e.g., murine) constant region cannot be formed, for example bytrans-switching between the transgene and endogenous Ig sequences. Usingembryonic stem cell technology and homologous recombination, theendogenous immunoglobulin repertoire can be readily eliminated. Inaddition, suppression of endogenous Ig genes may be accomplished using avariety of techniques, such as antisense technology.

In other aspects of the invention, it may be desirable to produce atrans-switched immunoglobulin. Antibodies comprising such chimerictrans-switched immunoglobulins can be used for a variety of applicationswhere it is desirable to have a non-human (e.g., murine) constantregion, e.g., for retention of effector functions in the host. Thepresence of a murine constant region can afford advantages over a humanconstant region, for example, to provide murine effector functions(e.g., ADCC, murine complement fixation) so that such a chimericantibody may be tested in a mouse disease model. Subsequent to theanimal testing, the human variable region encoding sequence may beisolated, e.g., by PCR™ amplification or cDNA cloning from the source(hybridoma clone), and spliced to a sequence encoding a desired humanconstant region to encode a human sequence antibody more suitable forhuman therapeutic use.

B6. Mutagenesis by PCR™

Site-specific mutagenesis is a technique useful in the preparation ofindividual antibodies through specific mutagenesis of the underlyingDNA. The technique further provides a ready ability to prepare and testsequence variants, incorporating one or more of the foregoingconsiderations, whether humanizing or not, by introducing one or morenucleotide sequence changes into the DNA.

Although many methods are suitable for use in mutagenesis, the use ofthe polymerase chain reaction (PCR™) is generally now preferred. Thistechnology offers a quick and efficient method for introducing desiredmutations into a given DNA sequence. The following text particularlydescribes the use of PCR™ to introduce point mutations into a sequence,as may be used to change the amino acid encoded by the given sequence.Adaptations of this method are also suitable for introducing restrictionenzyme sites into a DNA molecule.

In this method, synthetic oligonucleotides are designed to incorporate apoint mutation at one end of an amplified segment. Following PCR™, theamplified fragments are blunt-ended by treating with Klenow fragments,and the blunt-ended fragments are then ligated and subcloned into avector to facilitate sequence analysis.

To prepare the template DNA that one desires to mutagenize, the DNA issubcloned into a high copy number vector, such as pUC19, usingrestriction sites flanking the area to be mutated. Template DNA is thenprepared using a plasmid miniprep. Appropriate oligonucleotide primersthat are based upon the parent sequence, but which contain the desiredpoint mutation and which are flanked at the 5′ end by a restrictionenzyme site, are synthesized using an automated synthesizer. It isgenerally required that the primer be homologous to the template DNA forabout 15 bases or so. Primers may be purified by denaturingpolyacrylamide gel electrophoresis, although this is not absolutelynecessary for use in PCR™. The 5′ end of the oligonucleotides shouldthen be phosphorylated.

The template DNA should be amplified by PCR™, using the oligonucleotideprimers that contain the desired point mutations. The concentration ofMgCl₂ in the amplification buffer will generally be about 15 mM.Generally about 20-25 cycles of PCR™ should be carried out as follows:denaturation, 35 sec. at 95° C.; hybridization, 2 mM at 50° C.; andextension, 2 mM at 72° C. The PCR™ will generally include a last cycleextension of about 10 mM at 72° C. After the final extension step, about5 units of Klenow fragments should be added to the reaction mixture andincubated for a further 15 min. at about 30° C. The exonuclease activityof the Klenow fragments is required to make the ends flush and suitablefor blunt-end cloning.

The resultant reaction mixture should generally be analyzed bynondenaturing agarose or acrylamide gel electrophoresis to verify thatthe amplification has yielded the predicted product. One would thenprocess the reaction mixture by removing most of the mineral oils,extracting with chloroform to remove the remaining oil, extracting withbuffered phenol and then concentrating by precipitation with 100%ethanol. Next, one should digest about half of the amplified fragmentswith a restriction enzyme that cuts at the flanking sequences used inthe oligonucleotides. The digested fragments are purified on a lowgelling/melting agarose gel.

To subclone the fragments and to check the point mutation, one wouldsubclone the two amplified fragments into an appropriately digestedvector by blunt-end ligation. This would be used to transform E. coli,from which plasmid DNA could subsequently be prepared using a miniprep.The amplified portion of the plasmid DNA would then be analyzed by DNAsequencing to confirm that the correct point mutation was generated.This is important as Taq DNA polymerase can introduce additionalmutations into DNA fragments.

The introduction of a point mutation can also be effected usingsequential PCR™ steps. In this procedure, the two fragments encompassingthe mutation are annealed with each other and extended by mutuallyprimed synthesis. This fragment is then amplified by a second PCR™ step,thereby avoiding the blunt-end ligation required in the above protocol.In this method, the preparation of the template DNA, the generation ofthe oligonucleotide primers and the first PCR™ amplification areperformed as described above. In this process, however, the chosenoligonucleotides should be homologous to the template DNA for a stretchof between about 15 and about 20 bases and must also overlap with eachother by about 10 bases or more.

In the second PCR™ amplification, one would use each amplified fragmentand each flanking sequence primer and carry PCR™ for between about 20and about 25 cycles, using the conditions as described above. One wouldagain subclone the fragments and check that the point mutation wascorrect by using the steps outlined above.

In using either of the foregoing methods, it is generally preferred tointroduce the mutation by amplifying as small a fragment as possible. Ofcourse, parameters such as the melting temperature of theoligonucleotide, as will generally be influenced by the GC content andthe length of the oligo, should also be carefully considered. Theexecution of these methods, and their optimization if necessary, will beknown to those of skill in the art, and are further described in variouspublications, such as Current Protocols in Molecular Biology, 1995,incorporated herein by reference.

When performing site-specific mutagenesis, Table A can be employed as areference.

TABLE A Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAUB7. Antibody Fragments and Derivatives

Irrespective of the source of the original VEGFR2-blocking, humananti-VEGF antibody of the invention, either the intact antibody,antibody multimers, or any one of a variety of functional,antigen-binding regions of the antibody may be used in the presentinvention. Exemplary functional regions include antibody fragments thatcomprise an antigen binding domain such as Fab′, Fab, F(ab′)₂, singledomain antibodies (DABs), T and Abs dimer, Fv, scFv (single chain Fv),dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecificantibody fragments and the like. Techniques for preparing suchconstructs are well known to those in the art and are further describedherein.

The choice of antibody construct may be influenced by various factors.For example, prolonged half-life can result from the active readsorptionof intact antibodies within the kidney, a property of the Fc piece ofimmunoglobulin. IgG based antibodies, therefore, are expected to exhibitslower blood clearance than their Fab′ counterparts. However, Fab′fragment-based compositions will generally exhibit better tissuepenetrating capability.

If desired, particular Fc regions could be selected to providelongevity. For example, see WO 99/43713, which concerns constant domainswith enhanced circulating half-lives achieved by substantially reducedbinding to the Fcγ receptors, FcγRI, FcγRII and FcγRIII (Fridman, 1991).Additionally, U.S. Pat. No. 7,083,784 concerns modified constant domainswith increased in vivo half-lives resulting from modifications thatincrease their affinity for the FcRn (neonatal Fc receptor). Thetechniques of U.S. Pat. No. 7,083,784 may be applied to createantibodies with better longevity, either with or without substantialeffector functions.

Antibody fragments can be obtained by proteolysis of the whole humanimmunoglobulin by the non-specific thiol protease, papain. Papaindigestion yields two identical antigen-binding fragments, termed “Fabfragments”, each with a single antigen-binding site, and a residual “Fcfragment”.

Papain must first be activated by reducing the sulfhydryl group in theactive site with cysteine, 2-mercaptoethanol or dithiothreitol. Heavymetals in the stock enzyme should be removed by chelation with EDTA (2mM) to ensure maximum enzyme activity. Enzyme and substrate are normallymixed together in the ratio of 1:100 by weight. After incubation, thereaction can be stopped by irreversible alkylation of the thiol groupwith iodoacetamide or simply by dialysis. The completeness of thedigestion should be monitored by SDS-PAGE and the various fractionsseparated by protein A-Sepharose or ion exchange chromatography.

The usual procedure for preparation of F(ab′)₂ fragments from IgG ofhuman origin is limited proteolysis by the enzyme pepsin. Theconditions, 100× antibody excess w/w in acetate buffer at pH 4.5, 37°C., suggest that antibody is cleaved at the C-terminal side of theinter-heavy-chain disulfide bond. Rates of digestion of mouse IgG mayvary with subclass and conditions should be chosen to avoid significantamounts of completely degraded IgG. In particular, IgG_(2b) issusceptible to complete degradation. The other subclasses requiredifferent incubation conditions to produce optimal results, all of whichis known in the art.

Pepsin treatment of intact antibodies yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen. Exemplary conditions for digestion of IgG by pepsin requiresconditions including dialysis in 0.1 M acetate buffer, pH 4.5, and thenincubation for four hours with 1% w/w pepsin; IgG1 and IgG_(2a)digestion is improved if first dialyzed against 0.1 M formate buffer, pH2.8, at 4° C., for 16 hours followed by acetate buffer. IgG_(2b) givesmore consistent results with incubation in staphylococcal V8 protease(3% w/w) in 0.1 M sodium phosphate buffer, pH 7.8, for four hours at 37°C.

An Fab fragment also contains the constant domain of the light chain andthe first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. F(ab′)₂ antibody fragmentswere originally produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions (CDRs) of each variable domain interact to definean antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions (CDRs) conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three hypervariableregions (CDRs) specific for an antigen) has the ability to recognize andbind antigen.

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise theV_(H) and V_(L) domains of antibody, wherein these domains are presentin a single polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding.

The following patents are specifically incorporated herein by referencefor the purposes of even further supplementing the present teachingsregarding the preparation and use of functional, antigen-binding regionsof antibodies, including scFv, Fv, Fab′, Fab and F(ab′)₂ fragments ofthe anti-VEGF antibodies: U.S. Pat. Nos. 5,855,866; 5,965,132;6,051,230; 6,004,555; 5,877,289; and 6,093,399. WO 98/45331 is alsoincorporated herein by reference for purposes including even furtherdescribing and teaching the preparation of variable, hypervariable andcomplementarity determining (CDR) regions of antibodies.

“Diabodies” are small antibody fragments with two antigen-binding sites,which fragments comprise a heavy chain variable domain (V_(H)) connectedto a light chain variable domain (V_(L)) in the same polypeptide chain(V_(H)-V_(L)). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described in EP 404,097 and WO93/11161. “Linear antibodies”, which can be bispecific or monospecific,comprise a pair of tandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) thatform a pair of antigen binding regions, as described in Zapata et al.(1995).

In using a Fab′ or antigen binding fragment of an antibody, with theattendant benefits on tissue penetration, one may derive additionaladvantages from modifying the fragment to increase its half-life. Avariety of techniques may be employed, such as manipulation ormodification of the antibody molecule itself, and also conjugation toinert carriers. Any conjugation for the sole purpose of increasinghalf-life, rather than to deliver an agent to a target, should beapproached carefully in that Fab′ and other fragments are chosen topenetrate tissues. Nonetheless, conjugation to non-protein polymers,such PEG and the like, is contemplated.

Modifications other than conjugation are therefore based upon modifyingthe structure of the antibody fragment to render it more stable, and/orto reduce the rate of catabolism in the body. One mechanism for suchmodifications is the use of D-amino acids in place of L-amino acids.Those of ordinary skill in the art will understand that the introductionof such modifications needs to be followed by rigorous testing of theresultant molecule to ensure that it still retains the desiredbiological properties. Further stabilizing modifications include the useof the addition of stabilizing moieties to either the N-terminal or theC-terminal, or both, which is generally used to prolong the half-life ofbiological molecules. By way of example only, one may wish to modify thetermini by acylation or amination.

Moderate conjugation-type modifications for use with the presentinvention include incorporating a salvage receptor binding epitope intothe antibody fragment. Techniques for achieving this include mutation ofthe appropriate region of the antibody fragment or incorporating theepitope as a peptide tag that is attached to the antibody fragment. WO96/32478 is specifically incorporated herein by reference for thepurposes of further exemplifying such technology. Salvage receptorbinding epitopes are typically regions of three or more amino acids fromone or two loops of the Fc domain that are transferred to the analogousposition on the antibody fragment. The salvage receptor binding epitopesof WO 98/45331 are incorporated herein by reference for use with thepresent invention.

B8. Binding and Functional Assays

Although the present invention has significant utility in animal andhuman treatment regimens, it also has many other practical uses,including many in vitro uses. Certain of these uses are related to thespecific binding properties of the human antibodies or immunoconjugates.In that all the compounds of the invention include at least one VEGFbinding component, they may be used in virtually all of the bindingembodiments in which any anti-VEGF antibody may be used.

The presence of an attached agent, where relevant, although providingadvantageous properties, does not negate the utility of the humanantibody regions in any binding assay. Suitably useful binding assaysthus include those commonly employed in the art, such as in immunoblots,Western blots, dot blots, RIAs, ELISAs, immunohistochemistry,fluorescent activated cell sorting (FACS), immunoprecipitation, affinitychromatography, and the like, as further described herein.

Certain standard binding assays are those in which an antigen isimmobilized onto a solid support matrix, e.g., nitrocellulose, nylon ora combination thereof, such as in immunoblots, Western blots and relatedassays. Other important assays are ELISAs. All such assays may bereadily adapted for use in the detection of VEGF, as may be applied inthe diagnosis of an angiogenic disease. The agents of the invention mayalso be used in conjunction with both fresh-frozen and formalin-fixed,paraffin-embedded tissue blocks in immunohistochemistry; in fluorescentactivated cell sorting, flow cytometry or flow microfluorometry; inimmunoprecipitation; in antigen purification embodiments, such asaffinity chromatography, even including, in cases of bispecificantibodies, the one-step rapid purification of one or more antigens atthe same time; and in many other binding assays that will be known tothose of skill in the art given the information presented herein.

Further practical uses of the present human antibodies are as controlsin functional assays. These include many in vitro and ex vivo assays andsystems, as well as animal model studies. As the binding and functionalproperties of the human antibodies of the invention are particularlyspecific, i.e., they inhibit VEGF binding to and signaling via VEGFR2,but not VEGFR1, such “control” uses are actually extremely valuable. Theassays that benefit from such a practical application of the presentinvention include, for example, assays concerning VEGF-mediatedendothelial cell growth, VEGF-induced phosphorylation and VEGF-inducedvascular permeability, as well as the corneal micropocket assay ofneovascularization and the chick chorio-allantoic membrane assay (CAM)assay. These assays systems can also be developed into in vitro or exvivo drug screening assays, wherein the present provision of biologicalmaterials with well defined properties is particularly important.

C. Immunoconjugates

Although the present invention provides surprisingly effective naked orunconjugated human antibodies for use in anti-angiogenic methods,VEGFR2-blocking, human anti-VEGF antibody immunoconjugates, immunotoxinsand coaguligands are also provided hereby. Currently preferred agentsfor use in VEGFR2-blocking, human anti-VEGF antibody therapeuticconjugates are radiotherapeutic agents (as exemplified by theradiodiagnostics disclosed herein), chemotherapeutic agents,anti-angiogenic agents, apoptosis-inducing agents, anti-tubulin drugs,anti-cellular or cytotoxic agents, cytokines, chemokine, V-type ATPaseinhibitors and coagulants (coagulation factors).

To generate immunoconjugates, immunotoxins and coaguligands, recombinantexpression may be employed to create a fusion protein, as is known tothose of skill in the art and further disclosed herein. Equally,immunoconjugates, immunotoxins and coaguligands may be generated usingavidin:biotin bridges or any of the chemical conjugation andcross-linker technologies developed in reference to antibody conjugates.

C1. Toxic and Anti-Cellular Agents

For certain applications, the therapeutic agents will be cytotoxic orpharmacological agents, particularly cytotoxic, cytostatic or otherwiseanti-cellular agents having the ability to kill or suppress the growthor cell division of endothelial cells. In general, these aspects of theinvention contemplate the use of any pharmacological agent that can beconjugated to a VEGFR2-blocking, human anti-VEGF antibody of theinvention, and delivered in active form to the targeted endothelium.

Exemplary anti-cellular agents include chemotherapeutic agents, as wellas cytotoxins. Chemotherapeutic agents that may be used include:hormones, such as steroids; anti-metabolites, such as cytosinearabinoside, fluorouracil, methotrexate or aminopterin; anthracyclines;mitomycin C; vinca alkaloids; demecolcine; etoposide; mithramycin;anti-tumor alkylating agents, such as chlorambucil or melphalan. Otherembodiments may include agents such as cytokines. Basically, anyanti-cellular agent may be used, so long as it can be successfullyconjugated to, or associated with, an antibody in a manner that willallow its targeting, internalization, release and/or presentation toblood components at the site of the targeted endothelial cells.

There may be circumstances, such as when the target antigen does notinternalize by a route consistent with efficient intoxication by thetoxic compound, where one will desire to target chemotherapeutic agents,such as anti-tumor drugs, cytokines, antimetabolites, alkylating agents,hormones, and the like. A variety of chemotherapeutic and otherpharmacological agents have now been successfully conjugated toantibodies and shown to function pharmacologically, includingdoxorubicin, daunomycin, methotrexate, vinblastine, neocarzinostatin,macromycin, trenimon and α-amanitin.

In other circumstances, any potential side-effects from cytotoxin-basedtherapy may be eliminated by the use of DNA synthesis inhibitors, suchas daunorubicin, doxorubicin, adriamycin, and the like. These agents aretherefore preferred examples of anti-cellular agents for use in thepresent invention. In terms of cytostatic agents, such compoundsgenerally disturb the natural cell cycle of a target cell, preferably sothat the cell is taken out of the cell cycle.

A wide variety of cytotoxic agents are known that may be conjugated toVEGFR2-blocking, human anti-VEGF antibodies. Examples include numeroususeful plant-, fungus- or bacteria-derived toxins, which, by way ofexample, include various A chain toxins, particularly ricin A chain;ribosome inactivating proteins, such as saporin or gelonin; α-sarcin;aspergillin; restrictocin; ribonucleases, such as placentalribonuclease; diphtheria toxin; and pseudomonas exotoxin, to name just afew.

The well-known 1992 toxin book, “Genetically Engineered Toxins”, editedby Arthur E. Frankel, including the appendix, which includes the primaryamino acid sequences of a large number of toxins, is specificallyincorporated herein by reference, for purposes of further describing andenabling the use of toxins in targeted constructs.

Of the toxins, gelonin and ricin A chains are preferred. The mostpreferred toxin moiety for use herewith is toxin A chain that has beentreated to modify or remove carbohydrate residues, so-calleddeglycosylated A chain (dgA). Deglycosylated ricin A chain is preferredbecause of its extreme potency, longer half-life, and because it iseconomically feasible to manufacture it in a clinical grade and scale.

It may be desirable from a pharmacological standpoint to employ thesmallest molecule possible that nevertheless provides an appropriatebiological response. One may thus desire to employ smaller A chainpeptides that will provide an adequate anti-cellular response. To thisend, it has been discovered that ricin A chain may be “truncated” by theremoval of 30 N-terminal amino acids by Nagarase (Sigma), and stillretain an adequate toxin activity. It is proposed that where desired,this truncated A chain may be employed in conjugates in accordance withthe invention.

Alternatively, one may find that the application of recombinant DNAtechnology to the toxin A chain moiety will provide additional benefitsin accordance the invention. In that the cloning and expression ofbiologically active ricin A chain has been achieved, it is now possibleto identify and prepare smaller, or otherwise variant peptides, whichnevertheless exhibit an appropriate toxin activity. Moreover, the factthat ricin A chain has now been cloned allows the application ofsite-directed mutagenesis, through which one can readily prepare andscreen for A chain-derived peptides and obtain additional usefulmoieties for use in connection with the present invention.

C2. Coagulation Factors

The VEGFR2-blocking, human anti-VEGF antibody of the invention may belinked to a component that is capable of directly or indirectlystimulating coagulation, to form a coaguligand. Here, the antibodies maybe directly linked to the coagulant or coagulation factor, or may belinked to a second binding region that binds and then releases thecoagulant or coagulation factor. As used herein, the terms “coagulant”and “coagulation factor” are each used to refer to a component that iscapable of directly or indirectly stimulating coagulation underappropriate conditions, preferably when provided to a specific in vivoenvironment, such as the tumor vasculature.

Preferred coagulation factors are Tissue Factor compositions, such astruncated TF (tTF), dimeric, multimeric and mutant TF molecules.“Truncated TF” (tTF) refers to TF constructs that are renderedmembrane-binding deficient by removal of sufficient amino acid sequencesto effect this change in property. A “sufficient amount” in this contextis an amount of transmembrane amino acid sequence originally sufficientto enter the TF molecule in the membrane, or otherwise mediatefunctional membrane binding of the TF protein. The removal of such a“sufficient amount of transmembrane spanning sequence” therefore createsa truncated Tissue Factor protein or polypeptide deficient inphospholipid membrane binding capacity, such that the protein issubstantially a soluble protein that does not significantly bind tophospholipid membranes. Truncated TF thus substantially fails to convertFactor VII to Factor Vila in a standard TF assay, and yet retainsso-called catalytic activity including activating Factor X in thepresence of Factor VIIa.

U.S. Pat. No. 5,504,067 is specifically incorporated herein by referencefor the purposes of further describing such truncated Tissue Factorproteins. Preferably, the Tissue Factors for use in these aspects of thepresent invention will generally lack the transmembrane and cytosolicregions (amino acids 220-263) of the protein. However, there is no needfor the truncated TF molecules to be limited to molecules of the exactlength of 219 amino acids.

Tissue Factor compositions may also be useful as dimers. Any of thetruncated, mutated or other Tissue Factor constructs may be prepared ina dimeric form for use in the present invention. As will be known tothose of ordinary skill in the art, such TF dimers may be prepared byemploying the standard techniques of molecular biology and recombinantexpression, in which two coding regions are prepared in-frame andexpressed from an expression vector. Equally, various chemicalconjugation technologies may be employed in connection with thepreparation of TF dimers. The individual TF monomers may be derivatizedprior to conjugation. All such techniques would be readily known tothose of skill in the art.

If desired, the Tissue Factor dimers or multimers may be joined via abiologically-releasable bond, such as a selectively-cleavable linker oramino acid sequence. For example, peptide linkers that include acleavage site for an enzyme preferentially located or active within atumor environment are contemplated. Exemplary forms of such peptidelinkers are those that are cleaved by urokinase, plasmin, thrombin,Factor IXa, Factor Xa, or a metalloproteinase, such as collagenase,gelatinase or stromelysin.

In certain embodiments, the Tissue Factor dimers may further comprise ahindered hydrophobic membrane insertion moiety, to later encourage thefunctional association of the Tissue Factor with the phospholipidmembrane, but only under certain defined conditions. As described in thecontext of the truncated Tissue Factors, hydrophobicmembrane-association sequences are generally stretches of amino acidsthat promote association with the phospholipid environment due to theirhydrophobic nature. Equally, fatty acids may be used to provide thepotential membrane insertion moiety.

Such membrane insertion sequences may be located either at theN-terminus or the C-terminus of the TF molecule, or generally appendedat any other point of the molecule so long as their attachment theretodoes not hinder the functional properties of the TF construct. Theintent of the hindered insertion moiety is that it remainsnon-functional until the TF construct localizes within the tumorenvironment, and allows the hydrophobic appendage to become accessibleand even further promote physical association with the membrane. Again,it is contemplated that biologically-releasable bonds andselectively-cleavable sequences will be particularly useful in thisregard, with the bond or sequence only being cleaved or otherwisemodified upon localization within the tumor environment and exposure toparticular enzymes or other bioactive molecules.

In other embodiments, the tTF constructs may be multimeric or polymeric.In this context a “polymeric construct” contains 3 or more Tissue Factorconstructs. A “multimeric or polymeric TF construct” is a construct thatcomprises a first TF molecule or derivative operatively attached to atleast a second and a third TF molecule or derivative. The multimers maycomprise between about 3 and about 20 such TF molecules. The individualTF units within the multimers or polymers may also be linked byselectively-cleavable peptide linkers or other biological-releasablebonds as desired. Again, as with the TF dimers discussed above, theconstructs may be readily made using either recombinant manipulation andexpression or using standard synthetic chemistry.

Even further TF constructs useful in context of the present inventionare those mutants deficient in the ability to activate Factor VII. Such“Factor VII activation mutants” are generally defined herein as TFmutants that bind functional Factor VII/VIIa, proteolytically activateFactor X, but are substantially free from the ability to proteolyticallyactivate Factor VII. Accordingly, such constructs are TF mutants thatlack Factor VII activation activity.

The ability of such Factor VII activation mutants to function inpromoting tumor-specific coagulation is based upon their specificdelivery to the tumor vasculature, and the presence of Factor VIIa atlow levels in plasma. Upon administration of such a Factor VIIactivation mutant conjugate, the mutant will be localized within thevasculature of a vascularized tumor. Prior to localization, the TFmutant would be generally unable to promote coagulation in any otherbody sites, on the basis of its inability to convert Factor VII toFactor VIIa. However, upon localization and accumulation within thetumor region, the mutant will then encounter sufficient Factor VIIa fromthe plasma in order to initiate the extrinsic coagulation pathway,leading to tumor-specific thrombosis. Exogenous Factor VIIa could alsobe administered to the patient.

Any one or more of a variety of Factor VII activation mutants may beprepared and used in connection with the present invention. There is asignificant amount of scientific knowledge concerning the recognitionsites on the TF molecule for Factor VII/VIIa. It will thus be understoodthat the Factor VII activation region generally lies between about aminoacid 157 and about amino acid 167 of the TF molecule. However, it iscontemplated that residues outside this region may also prove to berelevant to the Factor VII activating activity, and one may thereforeconsider introducing mutations into any one or more of the residuesgenerally located between about amino acid 106 and about amino acid 209of the TF sequence (WO 94/07515; WO 94/28017; each incorporated hereinby reference).

A variety of other coagulation factors may be used in connection withthe present invention, as exemplified by the agents set forth below.Thrombin, Factor V/Va and derivatives, Factor VIII/VIIIa andderivatives, Factor IX/IXa and derivatives, Factor X/Xa and derivatives,Factor XI/XIa and derivatives, Factor XII/XIIa and derivatives, FactorXIII/XIIIa and derivatives, Factor X activator and Factor V activatormay be used in the present invention.

Russell's viper venom Factor X activator is contemplated for use in thisinvention. Monoclonal antibodies specific for the Factor X activatorpresent in Russell's viper venom have also been produced, and could beused to specifically deliver the agent as part of a bispecific bindingligand.

Thromboxane A₂ is formed from endoperoxides by the sequential actions ofthe enzymes cyclooxygenase and thromboxane synthetase in plateletmicrosomes. Thromboxane A₂ is readily generated by platelets and is apotent vasoconstrictor, by virtue of its capacity to produce plateletaggregation. Both thromboxane A₂ and active analogues thereof arecontemplated for use in the present invention.

Thromboxane synthase, and other enzymes that synthesizeplatelet-activating prostaglandins, may also be used as “coagulants” inthe present context. Monoclonal antibodies to, and immunoaffinitypurification of, thromboxane synthase are known; as is the cDNA forhuman thromboxane synthase.

α2-antiplasmin, or α2-plasmin inhibitor, is a proteinase inhibitornaturally present in human plasma that functions to efficiently inhibitthe lysis of fibrin clots induced by plasminogen activator.α2-antiplasmin is a particularly potent inhibitor, and is contemplatedfor use in the present invention.

As the cDNA sequence for α2-antiplasmin is available, recombinantexpression and/or fusion proteins are preferred. Monoclonal antibodiesagainst α2-antiplasmin are also available that may be used in thebispecific binding ligand embodiments of the invention. These antibodiescould both be used to deliver exogenous α2-antiplasmin to the targetsite or to garner endogenous α2-antiplasmin and concentrate it withinthe targeted region.

C3. Anti-Tubulin Drugs

A range of drugs exert their effects via interfering with tubulinactivity. As tubulin functions are essential to mitosis and cellviability, certain “anti-tubulin drugs” are powerful chemotherapeuticagents. “Anti-tubulin drug(s)”, as used herein, means any agent, drug,prodrug or combination thereof that inhibits cell mitosis, preferably bydirectly or indirectly inhibiting tubulin activities necessary for cellmitosis, preferably tubulin polymerization or depolymerization.

Some of the more well known and currently preferred anti-tubulin drugsfor use with the present invention are colchicine; taxanes, such astaxol (paclitaxel) and docetaxel; vinca alkaloids, such as vinblastine,vincristine and vindescine; and combretastatins. Other suitableanti-tubulin drugs are cytochalasins (including B, J, E), dolastatin,auristatin PE, paclitaxel, ustiloxin D, rhizoxin, 1069C85, colcemid,albendazole, azatoxin and nocodazole.

As described in U.S. Pat. Nos. 5,892,069, 5,504,074 and 5,661,143,combretastatins are estradiol derivatives that generally inhibit cellmitosis. Exemplary combretastatins that may be used in conjunction withthe invention include those based upon combretastatin A, B and/or D andthose described in U.S. Pat. Nos. 5,892,069, 5,504,074 and 5,661,143.Combretastatins A-1, A-2, A-3, A-4, A-5, A-6, B-1, B-2, B-3 and B-4 areexemplary of the foregoing types.

U.S. Pat. Nos. 5,569,786 and 5,409,953 describe the isolation,structural characterization and synthesis of each of combretastatin A-1,A2, A-3, B-1, B-2, B-3 and B-4 and formulations and methods of usingsuch combretastatins to treat neoplastic growth. Any one or more of suchcombretastatins may be used in conjunction with the present invention.

Combretastatin A-4, as described in U.S. Pat. Nos. 5,892,069, 5,504,074,5,661,143 and 4,996,237, may also be used herewith. U.S. Pat. No.5,561,122 further describes suitable combretastatin A-4 prodrugs, whichare contemplated for combined use with the present invention.

U.S. Pat. No. 4,940,726 particularly describes macrocyclic lactonesdenominated combretastatin D-1 and ‘Combretastatin D-2’, each of whichmay be used in combination with the compositions and methods of thepresent invention. U.S. Pat. No. 5,430,062 concerns stilbene derivativesand combretastatin analogues with anti-cancer activity that may be usedin combination with the present invention.

C4. Anti-Angiogenic Agents

The present invention particularly provides combined anti-angiogenics.The human antibodies of the invention may be attached to an angiopoietin(Davis and Yancopoulos, 1999; Holash et al., 1999; incorporated hereinby reference), such as angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2),angiopoietin-3 (mouse) or angiopoietin-4 (human) (Valenzuela et al.,1999; Kim et al., 1999).

Exemplary anti-angiogenics for use herewith include angiostatin andendostatin. Angiostatin is disclosed in U.S. Pat. Nos. 5,776,704;5,639,725 and 5,733,876, each incorporated herein by reference.Angiostatin is a protein having a molecular weight of between about 38kD and about 45 kD, as determined by reducing polyacrylamide gelelectrophoresis, which contains approximately Kringle regions 1 through4 of a plasminogen molecule. Angiostatin generally has an amino acidsequence substantially similar to that of a fragment of murineplasminogen beginning at amino acid number 98 of an intact murineplasminogen molecule.

The amino acid sequence of angiostatin varies slightly between species.For example, in human angiostatin, the amino acid sequence issubstantially similar to the sequence of the above described murineplasminogen fragment, although an active human angiostatin sequence maystart at either amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. Further, human plasminogen may be used,as it has similar anti-angiogenic activity, as shown in a mouse tumormodel.

Angiostatin and endostatin have become the focus of intense study, asthey are the first angiogenesis inhibitors that have demonstrated theability to not only inhibit tumor growth but also cause tumorregressions in mice. There are multiple proteases that have been shownto produce angiostatin from plasminogen including elastase, macrophagemetalloelastase (MME), matrilysin (MMP-7), and 92 kDa gelatinase B/typeIV collagenase (MMP-9).

MME can produce angiostatin from plasminogen in tumors andgranulocyte-macrophage colony-stimulating factor (GMCSF) upregulates theexpression of MME by macrophages inducing the production of angiostatin.The role of MME in angiostatin generation is supported by the findingthat MME is in fact expressed in clinical samples of hepatocellularcarcinomas from patients. Another protease thought to be capable ofproducing angiostatin is stromelysin-1 (MMP-3). MMP-3 has been shown toproduce angiostatin-like fragments from plasminogen in vitro. Themechanism of action for angiostatin is currently unclear, it ishypothesized that it binds to an unidentified cell surface receptor onendothelial cells inducing endothelial cell to undergo programmed celldeath or mitotic arrest.

Endostatin appears to be an even more powerful anti-angiogenesis andanti-tumor agent and is particularly preferred for linking toVEGFR2-blocking, human anti-VEGF antibodies. Endostatin is effective atcausing regressions in a number of tumor models in mice. Tumors do notdevelop resistance to endostatin and, after multiple cycles oftreatment, tumors enter a dormant state during which they do notincrease in volume. In this dormant state, the percentage of tumor cellsundergoing apoptosis was increased, yielding a population thatessentially stays the same size.

U.S. Pat. No. 5,854,205, to Folkman and O'Reilly, specificallyincorporated herein by reference, concerns endostatin and its use as aninhibitor of endothelial cell proliferation and angiogenesis. Theendostatin protein corresponds to a C-terminal fragment of collagen typeXVIII, and the protein can be isolated from a variety of sources. U.S.Pat. No. 5,854,205 also teaches that endostatin can have an amino acidsequence of a fragment of collagen type XVIII, a collagen type XV, orBOVMPE 1 pregastric esterase. Combinations of endostatin with otheranti-angiogenic proteins, particularly angiostatin, are also describedby U.S. Pat. No. 5,854,205, such that the combined compositions arecapable of effectively regressing the mass of an angiogenesis-dependenttumor.

Endostatin and angiostatin, particularly endostatin, are preferredagents for tumor delivery according to the present invention.Vasculostatin, canstatin and maspin are also preferred agents.Endostatin fusion proteins may be prepared, as described in U.S. Pat.No. 6,342,221, incorporated herein by reference. Various forms ofchemically linked endostatin constructs may also be prepared, again asexemplified in U.S. Pat. No. 6,342,221.

C5. Apoptosis-Inducing Agents

The present invention may also be used to deliver agents that induceapoptosis in any cells within the tumor, including tumor cells and tumorvascular endothelial cells. Although many anti-cancer agents may have,as part of their mechanism of action, an apoptosis-inducing effect,certain agents have been discovered, designed or selected with this as aprimary mechanism, as described below.

Many forms of cancer have reports of mutations in tumor suppressorgenes, such as p53. Inactivation of p53 results in a failure to promoteapoptosis. With this failure, cancer cells progress in tumorigenesis,rather than become destined for cell death. Thus, delivery of tumorsuppressors is also contemplated for use in the present invention tostimulate cell death. Exemplary tumor suppressors include, but are notlimited to, p53, Retinoblastoma gene (Rb), Wilm's tumor (WT1), baxalpha, interleukin-1b-converting enzyme and family, MEN-1 gene,neurofibromatosis, type 1 (NF1), cdk inhibitor p16, colorectal cancergene (DCC), familial adenomatosis polyposis gene (FAP), multiple tumorsuppressor gene (MTS-1), BRCA1 and BRCA2.

Preferred for use are the p53 (U.S. Pat. Nos. 5,747,469; 5,677,178; and5,756,455; each incorporated herein by reference), Retinoblastoma, BRCA1(U.S. Pat. Nos. 5,750,400; 5,654,155; 5,710,001; 5,756,294; 5,709,999;5,693,473; 5,753,441; 5,622,829; and 5,747,282; each incorporated hereinby reference), MEN-1 (GenBank accession number U93236) and adenovirusE1A (U.S. Pat. No. 5,776,743; incorporated herein by reference) genes.

Other compositions that may be delivered by VEGFR2-blocking, humananti-VEGF antibodies include genes encoding the tumor necrosis factorrelated apoptosis inducing ligand termed TRAIL, and the TRAILpolypeptide (U.S. Pat. No. 5,763,223; incorporated herein by reference);the 24 kD apoptosis-associated protease of U.S. Pat. No. 5,605,826(incorporated herein by reference); Fas-associated factor 1, FAF1 (U.S.Pat. No. 5,750,653; incorporated herein by reference). Also contemplatedfor use in these aspects of the present invention is the provision ofinterleukin-1β-converting enzyme and family members, which are alsoreported to stimulate apoptosis.

Compounds such as carbostyril derivatives (U.S. Pat. Nos. 5,672,603; and5,464,833; each incorporated herein by reference); branched apogenicpeptides (U.S. Pat. No. 5,591,717; incorporated herein by reference);phosphotyrosine inhibitors and non-hydrolyzable phosphotyrosine analogs(U.S. Pat. Nos. 5,565,491; and 5,693,627; each incorporated herein byreference); agonists of RXR retinoid receptors (U.S. Pat. No. 5,399,586;incorporated herein by reference); and even antioxidants (U.S. Pat. No.5,571,523; incorporated herein by reference) may also be used. Tyrosinekinase inhibitors, such as genistein, may also be linked to the agentsof the present invention that target the cell surface receptor, VEGFR1(as supported by U.S. Pat. No. 5,587,459; incorporated herein byreference).

“Second mitochondrial-derived activator of caspase” (SMAC), also knownas DIABLO, is a protein that is released from the mitochondria duringapoptosis and binds to a family of proteins termed “inhibitor ofapoptosis proteins” (IAPs). IAP expression levels are increased in anumber of human tumors. Therefore, IAP antagonists or SMAC mimetics havebeen developed as anti-cancer agents. These may be used in conjunctionwith the present invention, both as conjugates and in combinationtherapies.

Exemplary IAP inhibitors include those developed on the basis of thecrystal structure of the interaction of SMAC with the BIR3 domain ofX-linked IAP (XIAP, also known as BIRC4) and monovalent and bivalent IAPantagonists designed using a structure-based approach (Vince et al.,2007; Varfolomeev et al., 2007). SMAC mimetics designed to resemble theN-terminal amino acids of SMAC, which interact with the BIR3 domain ofXIAP (Petersen et al., 2007), may also be used. It has been shown thatSMAC mimetics can induce regression of sensitive human lung cancerxenografts even as single agents, with 40% of treated animals remainingfree of tumors (Petersen et al., 2007).

C6. Cytokines

Cytokines and chemokines are particular examples of agents for linkingto a VEGFR2-blocking, human anti-VEGF antibody of the present invention.A range of cytokines may be used, including IL-3, IL-4, IL-5, IL-7,IL-8, IL-9, IL-11, IL-13, TGF-β, M-CSF, G-CSF, TNFβ, LAF, TCGF, BCGF,TRF, BAF, BDG, MP, LIF, OSM, TMF, IFN-α, IFN-β. More preferred cytokinesinclude IL-1α, IL-1β, IL-2, IL-6, IL-10, GM-CSF, IFN-γ, monocytechemoattractant protein-1 (MCP-1), platelet-derived growth factor-BB(PDGF-BB) and C-reactive protein (CRP) and the like. Particularlypreferred examples are TNFα, TNFα inducers, IL-2, IL-12, IFN-α, IFN-β,IFN-γ and LEC.

IL-12, for example, may be attached to a VEGFR2-blocking, humananti-VEGF antibody and used to redirect host defenses to attack thetumor vessels. The chemokine LEC (liver-expressed chemokine, also knownas NCC-4, HCC-4, or LMC) is another preferred component (Giovarelli etal., 2000). LEC is chemotactic for dendritic cells, monocytes, T cells,NK cells and neutrophils and can therefore improve host-mediatedanti-tumor responses.

C7. Biologically Functional Equivalents

Equivalents, or even improvements, of the VEGFR2-blocking, humananti-VEGF antibodies of the invention can now be made. Modifications andchanges may be made in the structure of such an antibody and stillobtain a molecule having like or otherwise desirable characteristics.For example, certain amino acids may substituted for other amino acidsin a protein structure without appreciable loss of interactive bindingcapacity. These considerations also apply to toxins, anti-angiogenicagents, apoptosis-inducing agents, coagulants and the like.

Since it is the interactive capacity and nature of a protein thatdefines that protein's biological functional activity, certain aminoacid sequence substitutions can be made in a protein sequence (or ofcourse, the underlying DNA sequence) and nevertheless obtain a proteinwith like (agonistic) properties. It is thus contemplated that variouschanges may be made in the sequence of the antibodies or therapeuticagents (or underlying DNA sequences) without appreciable loss of theirbiological utility or activity. Biological functional equivalents madefrom mutating an underlying DNA sequence can be made using the codoninformation provided herein in Table A, and the supporting technicaldetails on site-specific mutagenesis.

It also is well understood by the skilled artisan that, inherent in thedefinition of a “biologically functional equivalent” protein or peptide,is the concept that there is a limit to the number of changes that maybe made within a defined portion of the molecule and still result in amolecule with an acceptable level of equivalent biological activity.Biologically functional equivalent proteins and peptides are thusdefined herein as those proteins and peptides in which certain, not mostor all, of the amino acids may be substituted. Of course, a plurality ofdistinct proteins/peptides with different substitutions may easily bemade and used in accordance with the invention. Such “biologicallyfunctional equivalent” peptides may be regarded as further examples of“substantially homologous” sequences as described herein.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

In making more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics,these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte and Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is thus understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent protein. As detailed in U.S. Pat. No. 4,554,101 (incorporatedherein by reference), the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those which are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

C8. Fusion Proteins and Recombinant Expression

The VEGFR2-blocking, human anti-VEGF antibody or immunoconjugates of thepresent invention may be readily prepared as fusion proteins usingmolecular biological techniques. Any fusion protein may be designed andmade using any of the therapeutic agents disclosed herein and thoseknown in the art. The fusion protein technology is readily adapted toprepare fusion proteins in which the two portions are joined by aselectively cleavable peptide sequence. Any therapeutic agent may beattached to the terminus of the antibody or to any point distinct fromthe CDRs. Therapeutic agents may also be prepared “integrally”, whereinthey are preferably associated with a selectively cleavable peptide toallow release of the agent after targeting.

The use of recombinant DNA techniques to achieve such ends is nowstandard practice to those of skill in the art. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. DNA and RNA synthesismay, additionally, be performed using an automated synthesizers (see,for example, the techniques described in Sambrook et al., 1989;incorporated herein by reference).

The preparation of such a fusion protein generally entails thepreparation of a first and second DNA coding region and the functionalligation or joining of such regions, in frame, to prepare a singlecoding region that encodes the desired fusion protein. In the presentcontext, the VEGFR2-blocking, human anti-VEGF antibody DNA sequence willbe joined in frame with a DNA sequence encoding a therapeutic agent. Itis not generally believed to be particularly relevant which portion ofthe construct is prepared as the N-terminal region or as the C-terminalregion.

Once the desired coding region has been produced, an expression vectoris created. Expression vectors contain one or more promoters upstream ofthe inserted DNA regions that act to promote transcription of the DNAand to thus promote expression of the encoded recombinant protein. Thisis the meaning of “recombinant expression”. To obtain a so-called“recombinant” version of the VEGFR2-blocking, human anti-VEGF antibodyof the invention or immunoconjugate thereof, it is expressed in arecombinant cell.

The engineering of DNA segment(s) for expression in a prokaryotic oreukaryotic system may be performed by techniques generally known tothose of skill in recombinant expression. It is believed that virtuallyany expression system may be employed in the expression of aVEGFR2-blocking, human anti-VEGF antibody or immunoconjugate constructs.

Such proteins may be successfully expressed in eukaryotic expressionsystems, e.g., CHO cells, however, it is envisioned that bacterialexpression systems, such as E. coli pQE-60 will be particularly usefulfor the large-scale preparation and subsequent purification of theVEGFR2-blocking, human anti-VEGF antibody or immunoconjugates. cDNAs mayalso be expressed in bacterial systems, with the encoded proteins beingexpressed as fusions with α-galactosidase, ubiquitin, Schistosomajaponicum glutathione S-transferase, and the like. It is believed thatbacterial expression will have advantages over eukaryotic expression interms of ease of use and quantity of materials obtained thereby.

In terms of microbial expression, U.S. Pat. Nos. 5,583,013; 5,221,619;4,785,420; 4,704,362; and 4,366,246 are incorporated herein by referencefor the purposes of even further supplementing the present disclosure inconnection with the expression of genes in recombinant host cells.

Recombinantly produced VEGFR2-blocking, human anti-VEGF antibodiesimmunoconjugates may be purified and formulated for humanadministration. Alternatively, nucleic acids encoding theimmunoconjugates may be delivered via gene therapy. Although nakedrecombinant DNA or plasmids may be employed, the use of liposomes orvectors is preferred. The ability of certain viruses to enter cells viareceptor-mediated endocytosis and to integrate into the host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells.Preferred gene therapy vectors for use in the present invention willgenerally be viral vectors.

Retroviruses have promise as gene delivery vectors due to their abilityto integrate their genes into the host genome, transferring a largeamount of foreign genetic material, infecting a broad spectrum ofspecies and cell types and of being packaged in special cell-lines.Other viruses, such as adenovirus, herpes simplex viruses (HSV),cytomegalovirus (CMV), and adeno-associated virus (AAV), such as thosedescribed by U.S. Pat. No. 5,139,941 (incorporated herein by reference),may also be engineered to serve as vectors for gene transfer.

Although some viruses that can accept foreign genetic material arelimited in the number of nucleotides they can accommodate and in therange of cells they infect, these viruses have been demonstrated tosuccessfully effect gene expression. However, adenoviruses do notintegrate their genetic material into the host genome and therefore donot require host replication for gene expression, making them ideallysuited for rapid, efficient, heterologous gene expression. Techniquesfor preparing replication-defective infective viruses are well known inthe art.

In certain further embodiments, the gene therapy vector will be HSV. Afactor that makes HSV an attractive vector is the size and organizationof the genome. Because HSV is large, incorporation of multiple genes orexpression cassettes is less problematic than in other smaller viralsystems. In addition, the availability of different viral controlsequences with varying performance (e.g., temporal, strength) makes itpossible to control expression to a greater extent than in othersystems. It also is an advantage that the virus has relatively fewspliced messages, further easing genetic manipulations. HSV also isrelatively easy to manipulate and can be grown to high titers.

Of course, in using viral delivery systems, one will desire to purifythe virion sufficiently to render it essentially free of undesirablecontaminants, such as defective interfering viral particles orendotoxins and other pyrogens such that it will not cause any untowardreactions in the cell, animal or individual receiving the vectorconstruct. A preferred means of purifying the vector involves the use ofbuoyant density gradients, such as cesium chloride gradientcentrifugation.

C9. Antibody Conjugates

VEGFR2-blocking, human anti-VEGF antibodies may be conjugated toanti-cellular or cytotoxic agents, to prepare “immunotoxins”; oroperatively associated with components that are capable of directly orindirectly stimulating coagulation, thus forming a “coaguligand”. Incoaguligands, the antibody may be directly linked to a direct orindirect coagulation factor, or may be linked to a second binding regionthat binds and then releases a direct or indirect coagulation factor.The ‘second binding region’ approach generally uses a coagulant-bindingantibody as a second binding region, thus resulting in a bispecificantibody construct. The preparation and use of bispecific antibodies ingeneral is well known in the art, and is further disclosed herein.

In the preparation of immunotoxins, coaguligands and bispecificantibodies, recombinant expression may be employed. The nucleic acidsequences encoding the chosen antibody are attached, in-frame, tonucleic acid sequences encoding the chosen toxin, coagulant, or secondbinding region to create an expression unit or vector. Recombinantexpression results in translation of the new nucleic acid, to yield thedesired protein product. Although antibody-encoding nucleic acids areemployed, rather than protein binding ligands, the recombinant approachis essentially the same as those described hereinabove.

Returning to conjugate technology, the preparation of immunotoxins isgenerally well known in the art. However, certain advantages may beachieved through the application of certain preferred technology, bothin the preparation of the immunotoxins and in their purification forsubsequent clinical administration. For example, while IgG basedimmunotoxins will typically exhibit better binding capability and slowerblood clearance than their Fab′ counterparts, Fab′ fragment-basedimmunotoxins will generally exhibit better tissue penetrating capabilityas compared to IgG based immunotoxins.

Additionally, while numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate the toxinmoiety to the VEGFR2-blocking, human anti-VEGF antibody, certain linkerswill generally be preferred over other linkers, based on differingpharmacological characteristics and capabilities. For example, linkersthat contain a disulfide bond that is sterically “hindered” are to bepreferred, due to their greater stability in vivo, thus preventingrelease of the toxin moiety prior to binding at the site of action.

A wide variety of cytotoxic agents are known that may be conjugated toVEGFR2-blocking, human anti-VEGF antibody, including plant-, fungus- andbacteria-derived toxins, such as ricin A chain or deglycosylated Achain. The cross-linking of a toxin A chain to an antibody, in certaincases, requires a cross-linker that presents disulfide functions. Thereason for this is unclear, but is likely due to a need for certaintoxin moieties to be readily releasable from the antibody once the agenthas “delivered” the toxin to the targeted cells.

Each type of cross-linker, as well as how the cross-linking isperformed, will tend to vary the pharmacodynamics of the resultantconjugate. Ultimately, in cases where a releasable toxin iscontemplated, one desires to have a conjugate that will remain intactunder conditions found everywhere in the body except the intended siteof action, at which point it is desirable that the conjugate have good“release” characteristics. Therefore, the particular cross-linkingscheme, including in particular the particular cross-linking reagentused and the structures that are cross-linked, will be of somesignificance.

Depending on the specific toxin compound used as part of the fusionprotein, it may be necessary to provide a peptide spacer operativelyattaching the antibody and the toxin compound that is capable of foldinginto a disulfide-bonded loop structure. Proteolytic cleavage within theloop would then yield a heterodimeric polypeptide wherein the antibodyand the toxin compound are linked by only a single disulfide bond. Anexample of such a toxin is a Ricin A-chain toxin.

When certain other toxin compounds are utilized, a non-cleavable peptidespacer may be provided to operatively attach the VEGFR2-blocking, humananti-VEGF antibody and the toxin compound of the fusion protein. Toxinsthat may be used in conjunction with non-cleavable peptide spacers arethose which may, themselves, be converted by proteolytic cleavage, intoa cytotoxic disulfide-bonded form. An example of such a toxin compoundis a Pseudomonas exotoxin compound.

There may be circumstances, such as when the target antigen does notinternalize by a route consistent with efficient intoxication byimmunotoxins, where one will desire to target chemotherapeutic agentssuch as anti-tumor drugs, other cytokines, antimetabolites, alkylatingagents, hormones, and the like. A variety of chemotherapeutic and otherpharmacological agents have now been successfully conjugated toantibodies and shown to function pharmacologically. Exemplaryantineoplastic agents that have been investigated include doxorubicin,daunomycin, methotrexate, vinblastine, and various others. Moreover, theattachment of other agents such as neocarzinostatin, macromycin,trenimon and α-amanitin has been described.

Where coagulation factors are used in connection with the presentinvention, any covalent linkage to the antibody should be made at a sitedistinct from its functional coagulating site. The compositions are thus“linked” in any operative manner that allows each region to perform itsintended function without significant impairment. Thus, the antibodybinds to VEGF, and the coagulation factor promotes blood clotting.

C10. Biochemical Cross-linkers

In additional to the general information provided above,VEGFR2-blocking, human anti-VEGF antibodies may be conjugated to one ormore therapeutic agents using certain preferred biochemicalcross-linkers. Cross-linking reagents are used to form molecular bridgesthat tie together functional groups of two different molecules. To linktwo different proteins in a step-wise manner, hetero-bifunctionalcross-linkers can be used that eliminate unwanted homopolymer formation.Exemplary hetero-bifunctional cross-linkers are referenced in Table B.

TABLE B HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Reactive Advantagesand Length after Linker Toward Applications cross-linking SMPT Primaryamines Greater stability 11.2 A Sulfhydryls SPDP Primary aminesThiolation 6.8 A Sulfhydryls Cleavable cross-linking LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Sulfo-LC- Primary aminesExtended spacer arm 15.6 A SPDP Sulfhydryls Water-soluble SMCC Primaryamines Stable maleimide 11.6 A Sulfhydryls reactive groupEnzyme-antibody conjugation Hapten-carrier protein conjugation Sulfo-Primary amines Stable maleimide 11.6 A SMCC Sulfhydryls reactive groupWater-soluble Enzyme-antibody conjugation MBS Primary aminesEnzyme-antibody 9.9 A Sulfhydryls conjugation Hapten-carrier proteinconjugation Sulfo-MBS Primary amines Water-soluble 9.9 A SulfhydrylsSIAB Primary amines Enzyme-antibody 10.6 A Sulfhydryls conjugationSulfo- Primary amines Water-soluble 10.6 A SIAB Sulfhydryls SMPB Primaryamines Extended spacer arm 14.5 A Sulfhydryls Enzyme-antibodyconjugation Sulfo- Primary amines Extended spacer arm 14.5 A SMPBSulfhydryls Water-soluble EDC/Sulfo- Primary amines Hapten-Carrier 0 NHSCarboxyl groups conjugation ABH Carbohydrates Reacts with sugar 11.9 ANonselective groups

Hetero-bifunctional cross-linkers contain two reactive groups: onegenerally reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other generally reacting with a thiol group (e.g.,pyridyl disulfide, maleimides, halogens, etc.). Through the primaryamine reactive group, the cross-linker may react with the lysineresidue(s) of one protein (e.g., the selected antibody or fragment) andthrough the thiol reactive group, the cross-linker, already tied up tothe first protein, reacts with the cysteine residue (free sulfhydrylgroup) of the other protein (e.g., the coagulant).

Compositions therefore generally have, or are derivatized to have, afunctional group available for cross-linking purposes. This requirementis not considered to be limiting in that a wide variety of groups can beused in this manner. For example, primary or secondary amine groups,hydrazide or hydrazine groups, carboxyl alcohol, phosphate, oralkylating groups may be used for binding or cross-linking

The spacer arm between the two reactive groups of a cross-linker mayhave various length and chemical compositions. A longer spacer armallows a better flexibility of the conjugate components while someparticular components in the bridge (e.g., benzene group) may lend extrastability to the reactive group or an increased resistance of thechemical link to the action of various aspects (e.g., disulfide bondresistant to reducing agents). The use of peptide spacers, such asL-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate antibodies andtoxic or coagulating agents. Linkers that contain a disulfide bond thatis sterically hindered may prove to give greater stability in vivo,preventing release of the agent prior to binding at the site of action.These linkers are thus one preferred group of linking agents.

One of the most preferred cross-linking reagents for use in immunotoxinsis SMPT, which is a bifunctional cross-linker containing a disulfidebond that is “sterically hindered” by an adjacent benzene ring andmethyl groups. It is believed that steric hindrance of the disulfidebond serves a function of protecting the bond from attack by thiolateanions such as glutathione which can be present in tissues and blood,and thereby help in preventing decoupling of the conjugate prior to thedelivery of the attached agent to the tumor site. It is contemplatedthat the SMPT agent may also be used in connection with the bispecificligands of this invention.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers can also beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane. The use of such cross-linkers is wellunderstood in the art.

Once conjugated, the conjugate is separated from unconjugated targetingand therapeutic agents and from other contaminants. A large a number ofpurification techniques are available for use in providing conjugates ofa sufficient degree of purity to render them clinically useful.Purification methods based upon size separation, such as gel filtration,gel permeation or high performance liquid chromatography, will generallybe of most use. Other chromatographic techniques, such as Blue-Sepharoseseparation, may also be used.

C11. Biologically Releasable Linkers

Although it is preferred that any linking moiety will have reasonablestability in blood, to prevent substantial release of the attached agentbefore targeting to the disease or tumor site, in certain aspects, theuse of biologically-releasable bonds and/or selectively cleavablespacers or linkers is contemplated. “Biologically-releasable bonds” and“selectively cleavable spacers or linkers” still have reasonablestability in the circulation.

The VEGFR2-blocking, human anti-VEGF antibodies of the present inventionmay thus be linked to one or more therapeutic agents via abiologically-releasable bond. Any form of VEGFR2-blocking, humananti-VEGF antibody may be employed, including intact antibodies,although ScFv fragments will be preferred in certain embodiments.

“Biologically-releasable bonds” or “selectively hydrolyzable bonds”include all linkages that are releasable, cleavable or hydrolyzable onlyor preferentially under certain conditions. This includes disulfide andtrisulfide bonds and acid-labile bonds, as described in U.S. Pat. Nos.5,474,765 and 5,762,918, each specifically incorporated herein byreference.

The use of an acid sensitive spacer for attachment of a therapeuticagent or drug to an antibody of the invention is particularlycontemplated. In such embodiments, the therapeutic agents or drugs arereleased within the acidic compartments inside a cell. It iscontemplated that acid-sensitive release may occur extracellularly, butstill after specific targeting, preferably to the tumor site. Certaincurrently preferred examples include human antibodies linked tocolchicine or doxorubicin via an acid sensitive spacer. Attachment viathe carbohydrate moieties of the antibodies is also contemplated. Insuch embodiments, the therapeutic agents or drugs are released withinthe acidic compartments inside a cell.

The human anti-VEGF antibody may also be derivatized to introducefunctional groups permitting the attachment of the therapeutic agent(s)through a biologically releasable bond. The human antibody may thus bederivatized to introduce side chains terminating in hydrazide,hydrazine, primary amine or secondary amine groups. Therapeutic agentsmay be conjugated through a Schiff's base linkage, a hydrazone or acylhydrazone bond or a hydrazide linker (U.S. Pat. Nos. 5,474,765 and5,762,918, each specifically incorporated herein by reference).

Also as described in U.S. Pat. Nos. 5,474,765 and 5,762,918, eachspecifically incorporated herein by reference, the human anti-VEGFantibody may be operatively attached to the therapeutic agent(s) throughone or more biologically releasable bonds that are enzyme-sensitivebonds, including peptide bonds, esters, amides, phosphodiesters andglycosides.

Preferred aspects of the invention concern the use of peptide linkersthat include at least a first cleavage site for a peptidase and/orproteinase that is preferentially located within a disease site,particularly within the tumor environment. The antibody-mediateddelivery of the attached therapeutic agent thus results in cleavagespecifically within the disease site or tumor environment, resulting inthe specific release of the active agent. Certain peptide linkers willinclude a cleavage site that is recognized by one or more enzymesinvolved in remodeling.

Peptide linkers that include a cleavage site for urokinase,pro-urokinase, plasmin, plasminogen, TGFβ, staphylokinase, Thrombin,Factor IXa, Factor Xa or a metalloproteinase, such as an interstitialcollagenase, a gelatinase or a stromelysin, are particularly preferred.U.S. Pat. Nos. 6,004,555, 5,877,289, and 6,093,399, are specificallyincorporated herein by reference for the purpose of further describingand enabling how to make and use targeting agent-therapeutic agentconstructs comprising biologically-releasable bonds andselectively-cleavable linkers and peptides. U.S. Pat. Nos. 5,877,289 and6,342,221, in particular, are specifically incorporated herein byreference for the purpose of further describing and enabling how to makeand use antibody constructs that comprise a selectively-cleavablepeptide linker that is cleaved by urokinase, plasmin, Thrombin, FactorIXa, Factor Xa or a metalloproteinase, such as an interstitialcollagenase, a gelatinase or a stromelysin, within a tumor environment.

Currently preferred selectively-cleavable peptide linkers are those thatinclude a cleavage site for plasmin or a metalloproteinase (also knownas “matrix metalloproteases” or “MMPs”), such as an interstitialcollagenase, a gelatinase or a stromelysin. Additional peptide linkersthat may be advantageously used in connection with the present inventioninclude, for example, the cleavable sequences from pro-urokinase, TGFβ,plasminogen, staphylokinase, Gelatinase A, various collagens, α₂M, PZP,α₁M, α₁I₃(2J) and α₁I₃(27J), including those particular sequencesdisclosed and claimed in U.S. Pat. No. 6,342,221, specificallyincorporated herein by reference.

C12. Bispecific Antibodies

Bispecific antibodies are particularly useful in the coaguligand andcombined anti-angiogenic aspects of the present invention. However,bispecific antibodies in general may be employed, so long as one armbinds to VEGF, and the bispecific antibody is attached to a therapeuticagent, generally at a site distinct from the antigen binding site.

In general, the preparation of bispecific antibodies is also well knownin the art. One method involves the separate preparation of antibodieshaving specificity for the targeted antigen, on the one hand, and (asherein) a coagulating agent on the other. Peptic F(ab′γ)₂ fragments areprepared from the two chosen antibodies, followed by reduction of eachto provide separate Fab′γ_(SH) fragments. The SH groups on one of thetwo partners to be coupled are then alkylated with a cross-linkingreagent such as o-phenylenedimaleimide to provide free maleimide groupson one partner. This partner may then be conjugated to the other bymeans of a thioether linkage, to give the desired F(ab′γ)₂heteroconjugate. Other techniques are known wherein cross-linking withSPDP or protein A is carried out, or a trispecific construct isprepared.

D. Pharmaceutical Compositions

The pharmaceutical compositions of the present invention will generallycomprise an effective amount of at least a first VEGFR2-blocking, humananti-VEGF antibody or immunoconjugate, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Combinedtherapeutics are also contemplated, and the same type of underlyingpharmaceutical compositions may be employed for both single and combinedmedicaments.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. Veterinary uses are equally included within theinvention and “pharmaceutically acceptable” formulations includeformulations for both clinical and/or veterinary use.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. For human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards. Supplementary activeingredients can also be incorporated into the compositions.

“Unit dosage” formulations are those containing a dose or sub-dose ofthe administered ingredient adapted for a particular timed delivery. Forexample, exemplary “unit dosage” formulations are those containing adaily dose or unit or daily sub-dose or a weekly dose or unit or weeklysub-dose and the like.

D1. Injectable Formulations

The VEGFR2-blocking, human anti-VEGF antibody antibodies orimmunoconjugates of the present invention will most often be formulatedfor parenteral administration, e.g., formulated for injection via theintravenous, intramuscular, sub-cutaneous, transdermal, or other suchroutes, including peristaltic administration and direct instillationinto a tumor or disease site (intracavity administration). Thepreparation of an aqueous composition that contains such an antibody orimmunoconjugate as an active ingredient will be known to those of skillin the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and fluid to theextent that syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

The VEGFR2-blocking, human anti-VEGF antibody or immunoconjugatecompositions can be formulated into a sterile aqueous composition in aneutral or salt form. Solutions as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Pharmaceutically acceptablesalts, include the acid addition salts (formed with the free aminogroups of the protein), and those that are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acids, or such organicacids as acetic, trifluoroacetic, oxalic, tartaric, mandelic, and thelike. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

Suitable carriers include solvents and dispersion media containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants.

Under ordinary conditions of storage and use, all such preparationsshould contain a preservative to prevent the growth of microorganisms.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Prior to or upon formulation, the VEGFR2-blocking, human anti-VEGFantibody or immunoconjugate should be extensively dialyzed to removeundesired small molecular weight molecules, and/or lyophilized for moreready formulation into a desired vehicle, where appropriate. Sterileinjectable solutions are prepared by incorporating the active agents inthe required amount in the appropriate solvent with various of the otheringredients enumerated above, as desired, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle thatcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above.

In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques that yield a powder of the active ingredient,plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the VEGFR2-blocking, human anti-VEGFantibody or immunoconjugate admixed with an acceptable pharmaceuticaldiluent or excipient, such as a sterile aqueous solution, to give arange of final concentrations, depending on the intended use. Thetechniques of preparation are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company, 1980, incorporated herein by reference. It should beappreciated that endotoxin contamination should be kept minimally at asafe level, for example, less that 0.5 ng/mg protein. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards. Upon formulation, the antibody or immunoconjugatesolutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

D2. Sustained Release Formulations

Formulations of VEGFR2-blocking, human anti-VEGF antibodies orimmunoconjugate solutions are easily administered in a variety of dosageforms, such as the type of injectable solutions described above, butother pharmaceutically acceptable forms are also contemplated, e.g.,tablets, pills, capsules or other solids for oral administration,suppositories, pessaries, nasal solutions or sprays, aerosols,inhalants, topical formulations, liposomal forms and the like. The typeof form for administration will be matched to the disease or disorder tobe treated.

Pharmaceutical “slow release” capsules or “sustained release”compositions or preparations may be used and are generally applicable.Slow release formulations are generally designed to give a constant druglevel over an extended period and may be used to deliver aVEGFR2-blocking, human anti-VEGF antibody or immunoconjugate inaccordance with the present invention. The slow release formulations aretypically implanted in the vicinity of the disease site, for example, atthe site of a tumor.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody or immunoconjugate, which matrices are in the form of shapedarticles, e.g., films or microcapsule. Examples of sustained-releasematrices include polyesters; hydrogels, for example,poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol); polylactides,e.g., U.S. Pat. No. 3,773,919; copolymers of L-glutamic acid and γethyl-L-glutamate; non-degradable ethylene-vinyl acetate; degradablelactic acid-glycolic acid copolymers, such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate); and poly-D-(−)-3-hydroxybutyric acid.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., thus reducing biologicalactivity and/or changing immunogenicity. Rational strategies areavailable for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism involves intermolecular S—S bondformation through thio-disulfide interchange, stabilization is achievedby modifying sulfhydryl residues, lyophilizing from acidic solutions,controlling moisture content, using appropriate additives, developingspecific polymer matrix compositions, and the like.

D3. Liposomes and Nanoparticles

In certain embodiments, liposomes and/or nanoparticles may also beemployed with the VEGFR2-blocking, human anti-VEGF antibodies orimmunoconjugates. The formation and use of liposomes is generally knownto those of skill in the art, as summarized below.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios, the liposome is the preferred structure. The physicalcharacteristics of liposomes depend on pH, ionic strength and thepresence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition that markedly alters their permeability. The phase transitioninvolves a change from a closely packed, ordered structure, known as thegel state, to a loosely packed, less-ordered structure, known as thefluid state. This occurs at a characteristic phase-transitiontemperature and results in an increase in permeability to ions, sugarsand drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

D4. Ophthalmic Formulations

Many diseases with an angiogenic component are associated with the eye.For example, diseases associated with corneal neovascularization thatcan be treated according to the present invention include, but are notlimited to, diabetic retinopathy, retinopathy of prematurity, cornealgraft rejection, neovascular glaucoma and retrolental fibroplasia,epidemic keratoconjunctivitis, Vitamin A deficiency, contact lensoverwear, atopic keratitis, superior limbic keratitis, pterygiumkeratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis,Mycobacteria infections, lipid degeneration, chemical burns, bacterialulcers, fungal ulcers, Herpes simplex infections, Herpes zosterinfections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, mariginal keratolysis, trauma,rheumatoid arthritis, systemic lupus, polyarteritis, Wegenerssarcoidosis, Scleritis, Steven's Johnson disease, periphigoid radialkeratotomy, and corneal graft rejection.

Diseases associated with retinal/choroidal neovascularization that canbe treated according to the present invention include, but are notlimited to, diabetic retinopathy, macular degeneration, sickle cellanemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease,vein occlusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales disease, Bechetsdisease, infections causing a retinitis or choroiditis, presumed ocularhistoplasmosis, Bests disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications.

Other diseases that can be treated according to the present inventioninclude, but are not limited to, diseases associated with rubeosis(neovascularization of the angle) and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue including all forms ofproliferative vitreoretinopathy, whether or not associated withdiabetes.

The VEGFR2-blocking, human anti-VEGF antibodies and immunoconjugates ofthe present invention may thus be advantageously employed in thepreparation of pharmaceutical compositions suitable for use asophthalmic solutions, including those for intravitreal and/orintracameral administration, either as a single agent or in combinationwith other ocular drugs or agents. For the treatment of any of theforegoing or other disorders a VEGFR2-blocking, human anti-VEGF antibodycomposition of the invention would be administered to the eye or eyes ofthe subject in need of treatment in the form of an ophthalmicpreparation prepared in accordance with conventional pharmaceuticalpractice, see for example “Remington's Pharmaceutical Sciences” 15thEdition, pages 1488 to 1501 (Mack Publishing Co., Easton, Pa.).

The ophthalmic preparation will contain at least a VEGFR2-blocking,human anti-VEGF antibody in a concentration from about 0.01 to about 1%by weight, preferably from about 0.05 to about 0.5% in apharmaceutically acceptable solution, suspension or ointment. Somevariation in concentration will necessarily occur, depending on theparticular compound employed, the condition of the subject to be treatedand the like, and the person responsible for treatment will determinethe most suitable concentration for the individual subject. Theophthalmic preparation will preferably be in the form of a sterileaqueous solution containing, if desired, additional ingredients, forexample preservatives, buffers, tonicity agents, antioxidants andstabilizers, nonionic wetting or clarifying agents, viscosity-increasingagents and the like.

Suitable preservatives for use in such a solution include benzalkoniumchloride, benzethonium chloride, chlorobutanol, thimerosal and the like.Suitable buffers include boric acid, sodium and potassium bicarbonate,sodium and potassium borates, sodium and potassium carbonate, sodiumacetate, sodium biphosphate and the like, in amounts sufficient tomaintain the pH at between about pH 6 and pH 8, and preferably, betweenabout pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran70, dextrose, glycerin, potassium chloride, propylene glycol, sodiumchloride, and the like, such that the sodium chloride equivalent of theophthalmic solution is in the range 0.9 plus or minus 0.2%.

Suitable antioxidants and stabilizers include sodium bisulfite, sodiummetabisulfite, sodium thiosulfite, thiourea and the like. Suitablewetting and clarifying agents include polysorbate 80, polysorbate 20,poloxamer 282 and tyloxapol. Suitable viscosity-increasing agentsinclude dextran 40, dextran 70, gelatin, glycerin,hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like. Theophthalmic preparation will be administered topically to the eye of thesubject in need of treatment by conventional methods, for example in theform of drops or by bathing the eye in the ophthalmic solution.

D5. Topical Formulations

In the broadest sense, formulations for topical administration includethose for delivery via the mouth (buccal) and through the skin “Topicaldelivery systems” also include transdermal patches containing theingredient to be administered. Delivery through the skin can further beachieved by iontophoresis or electrotransport, if desired.

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavored basis, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the ingredient to be administered in a suitableliquid carrier.

Formulations suitable for topical administration to the skin includeointments, creams, gels and pastes comprising the ingredient to beadministered in a pharmaceutical acceptable carrier. The formulation ofVEGFR2-blocking, human anti-VEGF antibodies for topical use, such as increams, ointments and gels, includes the preparation of oleaginous orwater-soluble ointment bases, as is well known to those in the art. Forexample, these compositions may include vegetable oils, animal fats, andmore preferably, semisolid hydrocarbons obtained from petroleum.Particular components used may include white ointment, yellow ointment,cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum, whitepetrolatum, spermaceti, starch glycerite, white wax, yellow wax,lanolin, anhydrous lanolin and glyceryl monostearate. Variouswater-soluble ointment bases may also be used, including glycol ethersand derivatives, polyethylene glycols, polyoxyl 40 stearate andpolysorbates.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate. Formulations suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing in addition to the active ingredient suchcarriers as are known in the art to be appropriate.

D6. Nasal Formulations

Local delivery via the nasal and respiratory routes is contemplated fortreating various conditions. These delivery routes are also suitable fordelivering agents into the systemic circulation. Formulations of activeingredients in carriers suitable for nasal administration are thereforealso included within the invention, for example, nasal solutions,sprays, aerosols and inhalants. Where the carrier is a solid, theformulations include a coarse powder having a particle size, forexample, in the range of 20 to 500 microns, which is administered, e.g.,by rapid inhalation through the nasal passage from a container of thepowder held close up to the nose.

Suitable formulations wherein the carrier is a liquid are useful innasal administration. Nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays andare prepared so that they are similar in many respects to nasalsecretions, so that normal ciliary action is maintained. Thus, theaqueous nasal solutions usually are isotonic and slightly buffered tomaintain a pH of 5.5 to 6.5. In addition, antimicrobial preservatives,similar to those used in ophthalmic preparations, and appropriate drugstabilizers, if required, may be included in the formulation. Variouscommercial nasal preparations are known and include, for example,antibiotics and antihistamines and are used for asthma prophylaxis.

Inhalations and inhalants are pharmaceutical preparations designed fordelivering a drug or compound into the respiratory tree of a patient. Avapor or mist is administered and reaches the affected area. This routecan also be employed to deliver agents into the systemic circulation.Inhalations may be administered by the nasal or oral respiratory routes.The administration of inhalation solutions is only effective if thedroplets are sufficiently fine and uniform in size so that the mistreaches the bronchioles.

Another group of products, also known as inhalations, and sometimescalled insufflations, comprises finely powdered or liquid drugs that arecarried into the respiratory passages by the use of special deliverysystems, such as pharmaceutical aerosols, that hold a solution orsuspension of the drug in a liquefied gas propellant. When releasedthrough a suitable valve and oral adapter, a metered does of theinhalation is propelled into the respiratory tract of the patient.Particle size is of major importance in the administration of this typeof preparation. It has been reported that the optimum particle size forpenetration into the pulmonary cavity is of the order of 0.5 to 7 μm.Fine mists are produced by pressurized aerosols and hence their use inconsidered advantageous.

E. Therapeutic Kits

This invention also provides therapeutic kits comprising aVEGFR2-blocking, human anti-VEGF antibody or immunoconjugate for use inthe present treatment methods. Such kits will generally contain, insuitable container means, a pharmaceutically acceptable formulation ofat least one VEGFR2-blocking, human anti-VEGF antibody orimmunoconjugate. The kits may also contain other pharmaceuticallyacceptable formulations, either for diagnosis/imaging or combinedtherapy. For example, such kits may contain any one or more of a rangeof chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents;anti-tumor cell antibodies; and/or anti-tumor vasculature or anti-tumorstroma immunotoxins or coaguligands.

The kits may have a single container (container means) that contains theVEGFR2-blocking, human anti-VEGF antibody or immunoconjugate, with orwithout any additional components, or they may have distinct containersfor each desired agent. Where combined therapeutics are provided, asingle solution may be pre-mixed, either in a molar equivalentcombination, or with one component in excess of the other.Alternatively, each of the VEGFR2-blocking, human anti-VEGF antibody orimmunoconjugate and other anti-cancer agent components of the kit may bemaintained separately within distinct containers prior to administrationto a patient.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is preferably an aqueous solution, with asterile aqueous solution being particularly preferred. However, thecomponents of the kit may be provided as dried powder(s). When reagentsor components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container.

The containers of the kit will generally include at least one vial, testtube, flask, bottle, syringe or other container means, into which theVEGFR2-blocking, human anti-VEGF antibody or immunoconjugate, and anyother desired agent, may be placed and, preferably, suitably aliquoted.Where separate components are included, the kit will also generallycontain a second vial or other container into which these are placed,enabling the administration of separated designed doses. The kits mayalso comprise a second/third container means for containing a sterile,pharmaceutically acceptable buffer or other diluent.

The kits may also contain a means by which to administer theVEGFR2-blocking, human anti-VEGF antibody or immunoconjugate to ananimal or patient, e.g., one or more needles or syringes, or even an eyedropper, pipette, or other such like apparatus, from which theformulation may be injected into the animal or applied to a diseasedarea of the body. The kits of the present invention will also typicallyinclude a means for containing the vials, or such like, and othercomponent, in close confinement for commercial sale, such as, e.g.,injection or blow-molded plastic containers into which the desired vialsand other apparatus are placed and retained.

F. Anti-Angiogenic Therapy

The present invention may be used to treat animals and patients withaberrant angiogenesis, such as that contributing to a variety ofdiseases and disorders, either alone or in combination therapies. Themost prevalent and/or clinically important of these, outside the fieldof cancer treatment, include arthritis, rheumatoid arthritis, psoriasis,atherosclerosis, diabetic retinopathy, age-related macular degeneration,Grave's disease, vascular restenosis, including restenosis followingangioplasty, arteriovenous malformations (AVM), meningioma, hemangiomaand neovascular glaucoma. Other potential targets for interventioninclude angiofibroma, atherosclerotic plaques, corneal graftneovascularization, hemophilic joints, hypertrophic scars, osler-webersyndrome, pyogenic granuloma retrolental fibroplasia, scleroderma,trachoma, vascular adhesions, synovitis, dermatitis, various otherinflammatory diseases and disorders, and even endometriosis. Furtherdiseases and disorders that are treatable by the invention, and theunifying basis of such angiogenic disorders, are set forth below.

One disease in which angiogenesis is involved is rheumatoid arthritis,wherein the blood vessels in the synovial lining of the joints undergoangiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis. Factors associatedwith angiogenesis also have a role in osteoarthritis, contributing tothe destruction of the joint.

Harada et al. (1998, specifically incorporated herein by reference)showed that VEGF is involved in the pathogenesis of rheumatoid arthritisand, furthermore, that measurement of serum concentration of VEGF is anoninvasive, useful method for monitoring the disease activity ofrheumatoid arthritis. This supports the therapeutic and diagnostic usesof the present invention in connection with rheumatoid arthritis.

Nagashima et al. (1999, specifically incorporated herein by reference)described the inhibitory effects of anti-rheumatic drugs on VEGF incultured rheumatoid synovial cells. VEGF is constitutively expressed inthe synovium of rheumatoid arthritis. The known anti-rheumatic drug,bucillamine (BUC), was shown to include within its mechanism of actionthe inhibition of VEGF production by synovial cells. Thus, theanti-rheumatic effects of BUC are mediated by suppression ofangiogenesis and synovial proliferation in the arthritic synoviumthrough the inhibition of VEGF production by synovial cells. The use ofthe present invention as an anti-arthritic therapy is supported by theVEGF inhibitory actions of this existing therapeutic.

Another example of a disease mediated by angiogenesis is ocularneovascular disease. This disease is characterized by invasion of newblood vessels into the structures of the eye, such as the retina orcornea. It is the most common cause of blindness and is involved inapproximately twenty eye diseases. In age-related macular degeneration,the associated visual problems are caused by an ingrowth of chorioidalcapillaries through defects in Bruch's membrane with proliferation offibrovascular tissue beneath the retinal pigment epithelium. Angiogenicdamage is also associated with diabetic retinopathy, retinopathy ofprematurity, corneal graft rejection, neovascular glaucoma andretrolental fibroplasia.

Other diseases associated with corneal neovascularization include, butare not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency,contact lens overwear, atopic keratitis, superior limbic keratitis,pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis,syphilis, Mycobacteria infections, lipid degeneration, chemical burns,bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpeszoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, mariginal keratolysis, rheumatoidarthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis,Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, andcorneal graph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,including age-related macular degeneration (AMD), sickle cell anemia,sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease, veinocclusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales disease, Bechetsdisease, infections causing a retinitis or choroiditis, presumed ocularhistoplasmosis, Bests disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications.

As to choroidal neovascularization, such as that associated with maculardegeneration, AMD and other ocular diseases, the VEGFR2-blocking, humananti-VEGF antibodies of the present invention are particularly wellsuited for use in treatment. This is, in part, because theysubstantially block VEGF binding to VEGFR2 without substantiallyblocking VEGF binding to VEGFR1 and the resultant benefits in the eye(Nozaki et al., 2006), which highlights another advantage of the presentinvention over existing anti-VEGF treatments, such as, e.g., Avastin andthe related product, Lucentis®.

Other diseases include, but are not limited to, diseases associated withrubeosis (neovascularization of the angle) and diseases caused by theabnormal proliferation of fibrovascular or fibrous tissue including allforms of proliferative vitreoretinopathy.

Chronic inflammation also involves pathological angiogenesis. Suchdisease states as ulcerative colitis and Crohn's disease showhistological changes with the ingrowth of new blood vessels into theinflamed tissues. Bartonellosis, a bacterial infection found in SouthAmerica, can result in a chronic stage that is characterized byproliferation of vascular endothelial cells.

Another pathological role associated with angiogenesis is found inatherosclerosis. The plaques formed within the lumen of blood vesselshave been shown to have angiogenic stimulatory activity. VEGF expressionin human coronary atherosclerotic lesions was demonstrated by Inoue etal. (1998, specifically incorporated herein by reference). Thisevidences the pathophysiological significance of VEGF in the progressionof human coronary atherosclerosis, as well as in recanalizationprocesses in obstructive coronary diseases. The present inventionprovides an effective treatment for such conditions.

One of the most frequent angiogenic diseases of childhood is thehemangioma. In most cases, the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, the hemangiomatoses, have a highmortality rate. Therapy-resistant hemangiomas exist that cannot betreated with therapeutics currently in use.

Angiogenesis is also responsible for damage found in hereditary diseasessuch as Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumors of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epistaxis(nosebleeds) or gastrointestinal bleeding and sometimes with pulmonaryor hepatic arteriovenous fistula.

Angiogenesis is also involved in normal physiological processes such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation or to prevent implantation by the blastula.

In wound healing, excessive repair or fibroplasia can be a detrimentalside effect of surgical procedures and may be caused or exacerbated byangiogenesis. Adhesions are a frequent complication of surgery and leadto problems such as small bowel obstruction.

Diseases and disorders characterized by undesirable vascularpermeability can also be treated by the present invention. These includeedema associated with brain tumors, ascites associated withmalignancies, Meigs' syndrome, lung inflammation, nephrotic syndrome,pericardial effusion and pleural effusion, as disclosed in WO 98/16551,specifically incorporated herein by reference.

Each of the foregoing diseases and disorders, along with all types oftumors, as described in the following sections, can be effectivelytreated by the present invention in accordance with the knowledge in theart, as disclosed in, e.g., U.S. Pat. No. 5,712,291 (specificallyincorporated herein by reference), that unified benefits result from theapplication of anti-angiogenic strategies to the treatment of angiogenicdiseases. Moreover, U.S. Pat. No. 6,524,583 is specifically incorporatedherein by reference for purposes including further describing andenabling the treatment of a wide-range of diseases and disorders usingan anti-VEGF antibody.

The human antibodies and/or immunoconjugates of the invention are mostpreferably utilized in the treatment of tumors. Tumors in whichangiogenesis is important include malignant tumors, and benign tumors,such as acoustic neuroma, neurofibroma, trachoma and pyogenicgranulomas. Angiogenesis is particularly prominent in solid tumorformation and metastasis. However, angiogenesis is also associated withblood-born tumors, such as leukemias, and various acute or chronicneoplastic diseases of the bone marrow in which unrestrainedproliferation of white blood cells occurs, usually accompanied byanemia, impaired blood clotting, and enlargement of the lymph nodes,liver, and spleen. Angiogenesis also plays a role in the abnormalitiesin the bone marrow that give rise to leukemia-like tumors.

Angiogenesis is important in two stages of tumor metastasis. In thevascularization of the primary tumor, angiogenesis allows cells to enterthe blood stream and to circulate throughout the body. After tumor cellshave left the primary site, and have settled into the secondary,metastasis site, angiogenesis must occur before the new tumor can growand expand. Therefore, prevention of angiogenesis can prevent metastasisof tumors and contain the neoplastic growth at the primary site,allowing treatment by other therapeutics, particularly, therapeuticagent-targeting agent constructs (see below).

The VEGFR2-blocking, human anti-VEGF antibody or immunoconjugate methodsprovided by this invention are thus broadly applicable to the treatmentof any malignant tumor having a vascular component. In using theantibodies and/or immunoconjugates of the invention in the treatment oftumors, particularly vascularized, malignant tumors, the agents may beused alone or in combination with, e.g., chemotherapeutic,radiotherapeutic, apoptopic, anti-angiogenic agents and/or immunotoxinsor coaguligands.

Typical vascularized tumors for treatment are the solid tumors,particularly carcinomas, which require a vascular component for theprovision of oxygen and nutrients. Exemplary solid tumors that may betreated using the invention include, but are not limited to, carcinomasof the lung, breast, ovary, stomach, pancreas, larynx, esophagus,testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus,endometrium, kidney, bladder, prostate, thyroid, squamous cellcarcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas,glioblastomas, neuroblastomas, and the like. WO 98/45331 is alsoincorporated herein by reference to further exemplify the variety oftumor types that may be effectively treated using an anti-VEGF antibody.

Knowledge of the role of angiogenesis in the maintenance and metastasisof tumors has led to a prognostic indicator for cancers such as breastcancer. The amount of neovascularization found in the primary tumor wasdetermined by counting the microvessel density in the area of the mostintense neovascularization in invasive breast carcinoma. A high level ofmicrovessel density was found to correlate with tumor recurrence.Control of angiogenesis by the therapies of the present invention willreduce or negate the recurrence of such tumors.

The present invention is contemplated for use in the treatment of anypatient that presents with a solid tumor. In light of the specificproperties of the VEGFR2-blocking, human anti-VEGF antibodycompositions, the therapeutics of the present invention will havereduced side effects. Particular advantages will result in themaintenance or enhancement of host immune responses against the tumor,and in the lack of adverse effects on bone tissue. The invention willthus be the anti-angiogenic therapy of choice for the treatment ofpediatric cancers and patients having, or at risk for developing,osteoporosis and other bone deficiencies.

Although all malignancies and solid tumors may be treated by theinvention, the unconjugated VEGFR2-blocking, human anti-VEGF antibodiesof this invention are particularly contemplated for use in treatingpatients with more angiogenic tumors, or patients at risk formetastasis.

The present invention is also intended as a preventative or prophylactictreatment. These aspects of the invention include the ability of theinvention to treat patients presenting with a primary tumor who may havemetastatic tumors, or tumor cells in the earlier stages of metastatictumor seeding. As an anti-angiogenic strategy, the present invention mayalso be used to prevent tumor development in subjects at moderate orhigh risk for developing a tumor, as based upon prognostic tests and/orclose relatives suffering from a hereditary cancer.

The conjugated or immunotoxin forms of the VEGFR2-blocking, humananti-VEGF antibodies of the invention are particularly contemplated foruse in destroying or de-bulking solid tumors. These aspects of theinvention may be used in conjunction with the unconjugatedanti-angiogenic antibodies of the invention, or with otheranti-angiogenic approaches.

It will be readily appreciated by those of skill in the art that theimmunoconjugate and prodrug forms of the present treatment methods havethe distinct advantage of providing a single therapeutic agent with twoproperties: the inherent anti-angiogenic property of the antibody andthe therapeutic property of the attached agent (e.g., cytotoxic,coagulative, apoptopic, etc). The conjugated and prodrug treatment formsof the present antibodies thus have an incredibly wide utilitythroughout the field of cancer treatment.

The guidance provided herein regarding the more suitable patients foruse in connection with the different aspects of the present invention isintended as teaching that certain patient's profiles may assist with theselection of patients for treatment by the present invention. Thepre-selection of certain patients, or categories of patients, does notin any way negate the usefulness of the present invention in connectionwith the treatment of all patients having a vascularized tumor, or otherangiogenic disease as described above. A further consideration is thefact that the assault on the tumor provided by the invention maypredispose the tumor to further therapeutic treatment, such that thesubsequent treatment results in an overall synergistic effect or evenleads to total remission or cure.

It is not believed that any particular type of tumor should be excludedfrom treatment using the present invention. However, the type of tumorcells may be relevant to the use of the invention in combination withother therapeutic agents, particularly chemotherapeutics and anti-tumorcell immunotoxins. Both the unconjugated and conjugated aspects of thepresent therapies will include an anti-angiogenic effect that willinhibit tumor vasculature proliferation. The conjugated and prodrugtreatment aspects will further destroy or occlude the tumor vasculature.As the vasculature is substantially or entirely the same in all solidtumors, the present methodology will be understood to be widely orentirely applicable to the treatment of all solid tumors, irrespectiveof the particular phenotype or genotype of the tumor cells themselves.

Therapeutically effective doses of VEGFR2-blocking, human anti-VEGFantibodies or immunoconjugate constructs are readily determinable usingdata from an animal model, e.g., as shown in the studies detailedherein. Experimental animals bearing solid tumors are frequently used tooptimize appropriate therapeutic doses prior to translating to aclinical environment. Such models are known to be very reliable inpredicting effective anti-cancer strategies. For example, mice bearingsolid tumors, such as used in the Examples, are widely used inpre-clinical testing. The inventors have used such art-accepted mousemodels to determine working ranges of therapeutic agents that givebeneficial anti-tumor effects with minimal toxicity.

In using unconjugated VEGFR2-blocking, human anti-VEGF antibodies inanti-angiogenic therapies, one can also draw on other published data inorder to assist in the formulation of doses for clinical treatment. Forinstance, although the antibodies of the present invention have distinctadvantages over those in the art, the information in the literatureconcerning treatment with other anti-VEGF antibodies can still be usedin combination with the data and teaching in the present application todesign and/or optimize treatment protocols and doses.

For example, Borgstrom et al. (1999), specifically incorporated hereinby reference, described the importance of VEGF in breast cancerangiogenesis in vivo using MAb A4.6.1. The humanized form of the A4.6.1antibody (Avastin, bevacizumab) has been approved for clinical use(Hurwitz et al., 2004). As the human antibodies of this inventionexhibited equivalent or even improved anti-tumor responses incomparative studies with A4.6.1/Avastin, these antibodies will also havesignificant utility in the treatment of cancer in humans, includingbreast cancer. The inventors further realized, as will be appreciated bythose of ordinary skill in the art, that patients with breast cancer aretypically women in the middle or later age groups, where concernsregarding osteoporosis are also apparent. The VEGFR2-blocking, humananti-VEGF antibodies of the present invention will thus have the addedadvantage of not causing an adverse effect on bone metabolism, and sowill be preferred for use in breast cancer patients having or at riskfor developing osteoporosis.

The same type of benefits make VEGFR2-blocking, human anti-VEGF antibodytherapeutics the preferred drugs for the treatment of pediatric cancers.In children with cancer, the need to continue healthy and substantialbone growth is evident. As VEGFR2-blocking, human anti-VEGF antibodieswill not substantially impair the activities of osteoclasts andchondroclasts, which are important in developing bone, these antibodieswill have important advantages over other antibodies, such as Avastin.

Borgstrom et al. (1999), specifically incorporated herein by reference,also reported that MAb A4.6.1 resulted in significant tumors regressionwhen used in combination with doxorubicin. This further supports thecombined use of VEGFR2-blocking, human anti-VEGF antibodies andconventional cytotoxic or chemotherapeutic agents to achieve significantclinical results in treating a variety of cancers. Both unconjugateddoxorubicin and doxorubicin prodrug combinations are contemplated.

Ferrara and colleagues also reported on the efficacy andconcentration-response of a murine anti-VEGF monoclonal antibody intumor-bearing mice and the extrapolation to human treatment (Mordenti etal., 1999, specifically incorporated herein by reference). The studieswere designed to evaluate the concentration-response relationship of themurine anti-VEGF monoclonal antibody so that an efficacious plasmaconcentration of the recombinant humanized form of the antibody could beestimated in cancer patients. Mordenti et al. (1999) concluded thatsatisfactory tumor suppression in nude mice was achieved using doses ofthe murine antibody that could be readily applied to the human system inorder to define clinical dosing regimens effective to maintain atherapeutic antibody for human use in the required efficacious range.Accordingly, the data from the present art-accepted mouse models canalso be translated into appropriate human doses using the type ofanalyses reported in Mordenti et al. (1999), in addition to thetechniques known to the skilled artisan as described herein.

Results from preclinical safety evaluations of a recombinant, humanizedform of Genentech's anti-VEGF antibody in monkeys (Ryan et al., 1999,specifically incorporated herein by reference) serve to exemplify thedrawbacks with that particular candidate therapeutic. Although theantibody has pharmacological activity in this animal, the monkeys inthese studies exhibited physeal dysplasia characterized by adose-related increase in hypertrophied chondrocytes, subchondral bonyplate formation, and inhibition of vascular invasion of the growthplate. No such drawbacks will be evident in the use of theVEGFR2-blocking, human anti-VEGF antibodies, which do not inhibit VEGFbinding and signaling in chondroclasts and chondrocytes, which ismediated by VEGFR1.

Data from a further study on the preclinical pharmacokinetics,interspecies scaling and tissue distribution of Genentech's humanizedmonoclonal anti-VEGF antibody was reported by Lin et al. (1999,specifically incorporated herein by reference). These studies wereconducted in mice, rats, monkeys and rabbits, the latter using¹²⁵I-labelled antibody. The pharmacokinetic data from mice, rats andmonkeys were used to predict the pharmacokinetics of the humanizedcounterpart antibody using allometric scaling in humans. Accordingly,appropriate dosage information can be developed for the treatment ofhuman pathological conditions, such as rheumatoid arthritis, ocularneovascularization and cancer.

The humanized version of the anti-VEGF antibody A4.6.1 (Avastin,bevacizumab) is now approved for clinical use (Hurwitz et al., 2004,incorporated herein by reference). Therefore, such clinical data canalso be considered as a reference source when designing therapeuticdoses for the present VEGFR2-blocking, human anti-VEGF antibodytreatment. The present invention shows the new human antibodies to be aseffective as A4.6.1/Avastin in studies in tumor-bearing mice, althoughthe specificity for inhibiting only VEGFR2-mediated actions of VEGF isan advantage. WO 98/45331 is also incorporated herein by reference tofurther exemplify the doses of humanized anti-VEGF antibodies that maybe used in treatment.

In terms of using conjugated VEGFR2-blocking, human anti-VEGF antibodiesin tumor therapy, one may refer to the scientific and patent literatureon the success of delivering a wide range of therapeutics to tumorvasculature to achieve a beneficial effect. By way of example, each ofU.S. Pat. Nos. 5,855,866; 5,877,289; 5,965,132; 6,051,230; 6,004,555;5,776,427; 6,004,554; 6,036,955; and 6,093,399 are incorporated hereinby reference for the purpose of further describing the use of suchtherapeutic agent-targeting agent constructs. In the present case, thetherapeutic agent-targeting agent constructs include targeting agentportions that exert an anti-angiogenic effect, which will magnify orotherwise enhance the anti-tumor activity of the attached therapeuticagent.

As is known in the art, there are realistic objectives that may be usedas a guideline in connection with pre-clinical testing before proceedingto clinical treatment. However, in light of the progress of otheranti-VEGF antibodies in the clinic, the demonstrated anti-tumor effectsin accepted models shown herein, and the enhanced safety of the presentstrategies, the current invention provides a therapeutic with a fasttrack to clinical treatment. Thus, pre-clinical testing may be employedto select the most advantageous antibodies, doses or combinations.

Any VEGFR2-blocking, human anti-VEGF antibody or immunoconjugate dose,or combined medicament, that results in any consistently detectableanti-angiogenic effect, inhibition of metastasis, tumor vasculaturedestruction, tumor thrombosis, necrosis and/or general anti-tumor effectwill define a useful invention. The present invention may also beeffective against vessels downstream of the tumor, i.e., target at leasta sub-set of the draining vessels, particularly as cytokines releasedfrom the tumor will be acting on these vessels, changing their antigenicprofile.

It will also be understood that even in such circumstances where theanti-angiogenic and/or tumor effects of the VEGFR2-blocking, humananti-VEGF antibody or immunoconjugate dose, or combined therapy, aretowards the low end of the intended therapeutic range, it may be thatthis therapy is still equally or even more effective than all otherknown therapies in the context of the particular tumor target orpatient. It is unfortunately evident to a clinician that certain tumorsand conditions cannot be effectively treated in the intermediate or longterm, but that does not negate the usefulness of the present therapy,particularly where it is at least about as effective as the otherstrategies generally proposed.

In designing appropriate doses of VEGFR2-blocking, human anti-VEGFantibody or immunoconjugate constructs, or combined therapeutics, forthe treatment of vascularized tumors, one may readily extrapolate fromthe animal studies described herein and the knowledge in the literaturein order to arrive at appropriate doses for clinical administration. Toachieve a conversion from animal to human doses, one would account forthe mass of the agents administered per unit mass of the experimentalanimal and, preferably, account for the differences in the body surfacearea (m²) between the experimental animal and the human patient. Allsuch calculations are well known and routine to those of ordinary skillin the art.

For example, taking the successful doses in the mouse studies, and byapplying standard calculations based upon mass and surface area,effective doses for use in human patients would be between about 1 mg/m²and about 1000 mg/m², preferably, between about 50 mg/m² and 500mg/m²10, and most preferably, between about 10 mg/m² and about 100mg/m². These doses are appropriate for VEGFR2-blocking, human anti-VEGFnaked antibodies and VEGFR2-blocking, human anti-VEGF immunoconjugates,although the doses are preferred for use in connection with naked orunconjugated antibodies for use as anti-angiogenics.

Accordingly, using this information, the inventors contemplate thatuseful low doses of VEGFR2-blocking, human anti-VEGF antibodies orimmunoconjugates for human administration will be about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45 or about 50 mg/m²; andthat useful high doses of such antibodies or immunoconjugates for humanadministration will be about 600, 650, 700, 750, 800, 850, 900, 925,950, 975 or about 1000 mg/m². Useful intermediate doses ofVEGFR2-blocking, human anti-VEGF antibodies or immunoconjugates forhuman administration are contemplated to be any dose between the low andhigh ranges, such as about 55, 60, 70, 80, 90, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, 500, 525, 550 or about 575 mg/m² or so.

Any particular range using any of the foregoing recited exemplary dosesor any value intermediate between the particular stated ranges iscontemplated. Where VEGFR2-blocking, human anti-VEGF immunoconjugatesare used, it will also be understood that coagulant immunoconjugates cangenerally be used at higher doses than toxin immunoconjugates.

In general, dosage ranges of between about 10-100 mg/m², about 10-90mg/m², about 10-80 mg/m², about 20-100 mg/m², about 20-90 mg/m², about20-80 mg/m², about 30-100 mg/m², about 30-90 mg/m², about 30-80 mg/m²,about 15-100 mg/m², about 25-100 mg/m², about 35-100 mg/m², about 15-90mg/m², about 25-90 mg/m², about 35-90 mg/m², or so of VEGFR2-blocking,human anti-VEGF antibodies or immunoconjugates will be preferred.Notwithstanding these stated ranges, it will be understood that, giventhe parameters and detailed guidance presented herein, furthervariations in the active or optimal ranges will be encompassed withinthe present invention.

Therefore, it will be understood that lower doses may be moreappropriate in combination with other agents, and that high doses canstill be tolerated, particularly given the enhanced safety of theVEGFR2-blocking, human anti-VEGF antibodies and immunoconjugates. Theuse of human antibodies (and optionally, human coagulant oranti-angiogenic proteins) renders the present invention even safer forclinical use, further reducing the chances of significant toxicity orside effects in healthy tissues.

The intention of the therapeutic regimens of the present invention isgenerally to produce significant anti-tumor effects whilst still keepingthe dose below the levels associated with unacceptable toxicity. Inaddition to varying the dose itself, the administration regimen can alsobe adapted to optimize the treatment strategy. One treatment protocol isto administer between about 1 mg/m² and about 1000 mg/m², preferably,between about 50 mg/m² and 500 mg/m²10, and most preferably, betweenabout 10 mg/m² and about 100 mg/m² of the VEGFR2-blocking, humananti-VEGF antibody or immunoconjugate, or therapeutic cocktailcontaining such, about 1 to 3 times a week, preferably by intravenous orintramuscular administration, and most preferably, intravenously.

In administering the particular doses, one would preferably provide apharmaceutically acceptable composition (according to FDA standards ofsterility, pyrogenicity, purity and general safety) to the patientsystemically. Intravenous injection is generally preferred. Continuousinfusion over a time period of about 1 or 2 hours or so is alsocontemplated.

Naturally, before wide-spread use, clinical trials will be conducted.The various elements of conducting a clinical trial, including patienttreatment and monitoring, will be known to those of skill in the art inlight of the present disclosure. The following information is beingpresented as a general guideline for use in establishing such trials.

Patients chosen for the first VEGFR2-blocking, human anti-VEGF antibodytreatment studies will have failed to respond to at least one course ofconventional therapy, and will have objectively measurable disease asdetermined by physical examination, laboratory techniques, and/orradiographic procedures. Any chemotherapy should be stopped at least 2weeks before entry into the study. Where murine monoclonal antibodies orantibody portions are employed, the patients should have no history ofallergy to mouse immunoglobulin.

Certain advantages will be found in the use of an indwelling centralvenous catheter with a triple lumen port. The VEGFR2-blocking, humananti-VEGF antibody should be filtered, for example, using a 0.22μfilter, and diluted appropriately, such as with saline, to a finalvolume of 100 ml. Before use, the test sample should also be filtered ina similar manner, and its concentration assessed before and afterfiltration by determining the A₂₈₀. The expected recovery should bewithin the range of 87% to 99%, and adjustments for protein loss canthen be accounted for.

The VEGFR2-blocking, human anti-VEGF antibodies or conjugates may beadministered over a period of approximately 4-24 hours, with eachpatient receiving 2-4 infusions at 2-7 day intervals. Administration canalso be performed by a steady rate of infusion over a 7 day period. Theinfusion given at any dose level should be dependent upon any toxicityobserved. Hence, if Grade II toxicity was reached after any singleinfusion, or at a particular period of time for a steady rate infusion,further doses should be withheld or the steady rate infusion stoppedunless toxicity improved. Increasing doses of VEGFR2-blocking, humananti-VEGF antibody should be administered to groups of patients untilapproximately 60% of patients showed unacceptable Grade III or IVtoxicity in any category. Doses that are 2/3 of this value are definedas the safe dose.

Physical examination, tumor measurements, and laboratory tests should,of course, be performed before treatment and at intervals up to 1 monthlater. Laboratory tests should include complete blood counts, serumcreatinine, creatine kinase, electrolytes, urea, nitrogen, SGOT,bilirubin, albumin, and total serum protein. Serum samples taken up to60 days after treatment should be evaluated by radioimmunoassay for thepresence of the administered therapeutic, and antibodies against anyportions thereof. Immunological analyses of sera, using any standardassay such as, for example, an ELISA or RIA, will allow thepharmacokinetics and clearance of the VEGFR2-blocking, human anti-VEGFantibody to be evaluated.

To evaluate the anti-tumor responses, the patients should be examined at48 hours to 1 week and again at 30 days after the last infusion. Whenpalpable disease was present, two perpendicular diameters of all massesshould be measured daily during treatment, within 1 week aftercompletion of therapy, and at 30 days. To measure nonpalpable disease,serial CT scans could be performed at 1-cm intervals throughout thechest, abdomen, and pelvis at 48 hours to 1 week and again at 30 days.Tissue samples should also be evaluated histologically, and/or by flowcytometry, using biopsies from the disease sites or even blood or fluidsamples if appropriate.

Clinical responses may be defined by acceptable measure. For example, acomplete response may be defined by the disappearance of all measurabletumor 1 month after treatment. Whereas a partial response may be definedby a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules 1 month aftertreatment, with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater 1 month aftertreatment, with progression in one or more sites.

In light of results from clinical trials, such as those described above,an even more precise treatment regimen may be formulated. Even so, somevariation in dosage may later be necessary depending on the condition ofthe subject being treated. The physician responsible for administrationwill, in light of the present disclosure, be able to determine theappropriate dose for the individual subject. Such optimization andadjustment is routinely carried out in the art and by no means reflectsan undue amount of experimentation.

G. Combination Therapies

Whether used for treating angiogenic diseases, such as arthritis,psoriasis, atherosclerosis, diabetic retinopathy, age-related maculardegeneration, Grave's disease, vascular restenosis, hemangioma andneovascular glaucoma (or other diseases described above), or solidtumors, the present invention can be combined with other therapies.

The VEGFR2-blocking, human anti-VEGF antibody treatment methods of thepresent invention may be combined with any other methods generallyemployed in the treatment of the particular tumor, disease or disorderthat the patient exhibits. So long as a particular therapeutic approachis not known to be detrimental to the patient's condition in itself, anddoes not significantly counteract the VEGFR2-blocking, human anti-VEGFantibody treatment, its combination with the present invention iscontemplated.

In connection solid tumor treatment, the present invention may be usedin combination with classical approaches, such as surgery, radiotherapy,chemotherapy, and the like. The invention therefore provides combinedtherapies in which VEGFR2-blocking, human anti-VEGF antibody constructsare used simultaneously with, before, or after surgery or radiationtreatment; or are administered to patients with, before, or afterconventional chemotherapeutic, radiotherapeutic or anti-angiogenicagents, or targeted immunotoxins or coaguligands.

The combined use of the invention with radiotherapy, radiotherapeutics,anti-angiogenic agents, apoptosis-inducing agents and anti-tubulin drugsis particularly preferred. Many examples of such agents have beendescribed above in conjunction with the immunoconjugates of the presentinvention. Any of the agents initially described for use as one part ofa therapeutic conjugate may also be used separately, but still inoperable combination with the present invention.

When one or more agents are used in combination with theVEGFR2-blocking, human anti-VEGF antibody therapy, there is norequirement for the combined results to be additive of the effectsobserved when each treatment is conducted separately. Although at leastadditive effects are generally desirable, any increased anti-tumoreffect above one of the single therapies would be of benefit. Also,there is no particular requirement for the combined treatment to exhibitsynergistic effects, although this is certainly possible andadvantageous.

To practice combined anti-angiogenic therapy, e.g., to treat an ocularor other angiogenic disease or disorder, one would simply administer toan animal a VEGFR2-blocking, human anti-VEGF antibody in combinationwith another therapeutic agent, including another (second)anti-angiogenic agent, in a manner effective to result in their combinedtherapeutic or anti-angiogenic actions within the animal. The agentswould therefore be provided in amounts effective and for periods of timeeffective to result in their combined presence within the disease siteand their combined actions in the disease environment, such as the eye.

To achieve this goal, the VEGFR2-blocking, human anti-VEGF antibody andother therapeutic or anti-angiogenic agent(s) may be administered to theanimal simultaneously, either in a single composition, or as twodistinct compositions using different administration routes.Alternatively, the VEGFR2-blocking, human anti-VEGF antibody treatmentmay precede, or follow, the other therapeutic or anti-angiogenictreatment by, e.g., intervals ranging from minutes to weeks and months.One would perform such treatment so that the VEGFR2-blocking, humananti-VEGF antibody and other therapeutic or anti-angiogenic agent(s)exert an advantageously combined therapeutic effect.

As to tumor therapy, to practice combined anti-tumor therapy, one wouldlikewise administer to an animal a VEGFR2-blocking, human anti-VEGFantibody in combination with another anti-cancer agent in a mannereffective to result in their combined anti-tumor actions within theanimal. The agents would again be provided in amounts effective and forperiods of time effective to result in their combined presence withinthe tumor vasculature and their combined actions in the tumorenvironment.

The VEGFR2-blocking, human anti-VEGF antibody and anti-cancer agents maybe administered to the animal simultaneously, either in a singlecomposition, or as two distinct compositions using differentadministration routes. Alternatively, the VEGFR2-blocking, humananti-VEGF antibody may be given before, or after, the anti-cancer agent,e.g., from minutes to weeks and months apart. The anti-cancer agent andVEGFR2-blocking, human anti-VEGF antibody would exert an advantageouslycombined effect on the tumor. Many anti-cancer agents would be givenprior to VEGFR2-blocking, human anti-VEGF antibody anti-angiogenictherapy. However, many other anti-cancer agents would be administeredsimultaneously with the VEGFR2-blocking, human anti-VEGF antibody orsubsequentlythereto, particularly when used after VEGFR2-blocking, humananti-VEGF immunoconjugates.

The general use of combinations of substances in cancer treatment iswell known. For example, U.S. Pat. No. 5,710,134 (incorporated herein byreference) discloses components that induce necrosis in tumors incombination with non-toxic substances or “prodrugs”. The enzymes setfree by necrotic processes cleave the non-toxic “prodrug” into the toxic“drug”, which leads to tumor cell death. Also, U.S. Pat. No. 5,747,469(incorporated herein by reference) discloses the combined use of viralvectors encoding p53 and DNA damaging agents. Any such similarapproaches can be used with the present invention.

In some situations, it may even be desirable to extend the time periodfor treatment significantly, where several days (2, 3, 4, 5, 6 or 7),several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even several months (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Thiswould be advantageous in circumstances where one treatment was intendedto substantially destroy the tumor, such as surgery or chemotherapy, andanother treatment was intended to prevent micrometastasis or tumorre-growth, such as anti-angiogenic based therapy. Anti-angiogenicsshould be administered at a careful time after surgery to alloweffective wound healing.

It also is envisioned that more than one administration of either theVEGFR2-blocking, human anti-VEGF antibody or the anti-cancer agent willbe utilized. The agents may be administered interchangeably, onalternate days or weeks; or a sequence of VEGFR2-blocking, humananti-VEGF antibody treatment may be given, followed by a sequence ofanti-cancer agent therapy. In any event, to achieve tumor regressionusing a combined therapy, all that is required is to deliver both agentsin a combined amount effective to exert an anti-tumor effect,irrespective of the times for administration.

In terms of surgery, any surgical intervention may be practiced incombination with the present invention. In connection with radiotherapy,any mechanism for inducing DNA damage locally within tumor cells iscontemplated, such as γ-irradiation, X-rays, UV-irradiation, microwavesand even electronic emissions and the like. The directed delivery ofradioisotopes to tumor cells is also contemplated, and this may be usedin connection with a targeting antibody or other targeting means, andpreferably, VEGFR2-blocking, human anti-VEGF antibodies.

Cytokine therapy also has proven to be an effective partner for combinedtherapeutic regimens. Various cytokines may be employed in such combinedapproaches. Examples of cytokines include IL-1α IL-1β, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF-β, GM-CSF,M-CSF, G-CSF, TNFα, TNFβ, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM,TMF, PDGF, IFN-α, IFN-13, IFN-γ. Cytokines are administered according tostandard regimens, consistent with clinical indications such as thecondition of the patient and relative toxicity of the cytokine.Uteroglobins may also be used to prevent or inhibit metastases (U.S.Pat. No. 5,696,092; incorporated herein by reference).

G1. Chemotherapeutics

In certain embodiments, the VEGFR2-blocking, human anti-VEGF antibodiesof the present invention may be administered in combination with achemotherapeutic agent. A variety of chemotherapeutic agents may be usedin the combined treatment methods disclosed herein. Chemotherapeuticagents contemplated as exemplary include, e.g., adriamycin,dactinomycin, mitomycin, caminomycin, daunomycin, doxorubicin,tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine,etoposide (VP-16), 5-fluorouracil (5FU), cytosine arabinoside,cyclophohphamide, thiotepa, methotrexate, camptothecin, actinomycin-D,mitomycin C, cisplatin (CDDP), aminopterin, combretastatin(s) andderivatives and prodrugs thereof.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. By way of example only, agents such as cisplatin, andother DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Further useful agents include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of polynucleotideprecursors may also be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorouracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU, is applicable in a widerange of carriers, including topical, however intravenous administrationwith doses ranging from 3 to 15 mg/kg/day being commonly used.

Exemplary chemotherapeutic agents for combined therapy are listed inTable C. Each of the agents listed are exemplary and not limiting. Theskilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Variation indosage will likely occur depending on the condition being treated. Thephysician administering treatment will be able to determine theappropriate dose for the individual subject.

TABLE C CHEMOTHERAPEUTIC AGENTS USEFUL IN NEOPLASTIC DISEASE CLASS TYPEOF AGENT EXAMPLES DISEASE Alkylating Nitrogen Mustards MechlorethamineHodgkin's disease, non-Hodgkin's Agents (chlormethine, lymphomasmustine, nitrogen mustard, HN₂) Mustargen ® Cyclophosphamide Acute andchronic lymphocytic (cytophosphane) leukemias, Hodgkin's disease, non-Cytoxan ®, Neosar ®, Hodgkin's lymphomas, multiple Revimmune ® myeloma,neuroblastoma, breast, ovary, lung, Wilms' tumor, cervix, testis,soft-tissue sarcomas Ifosfamide Non-Hodgkin's lymphomas, softMitoxana ®, Ifex ® tissue sarcoma, osteogenic sarcoma, testicular,breast, lung, cervical, ovarian, bone Melphalan Multiple myeloma,breast, ovary, (L-sarcolysin) melanoma Alkeran ® Chlorambucil Chroniclymphocytic leukemia, Leukeran ® primary macroglobulinemia, Hodgkin'sdisease, non-Hodgkin's lymphomas, ovarian Ethylenimenes andHexamethylmelamine Ovary Methylmelamines (Altretamine, HMM) Hexalen ®ThioTEPA Bladder, breast, ovary Alkyl Sulfonates Busulfan Chronicgranulocytic leukemia Myleran ®, Busulfex ® Nitrosoureas CarmustineHodgkin's disease, non-Hodgkin's BiCNU ® lymphomas, primary braintumors, multiple myeloma, malignant melanoma, glioma, glioblastomamultiforme, medulloblastoma, astrocytoma Lomustine (CCNU) Hodgkin'sdisease, non-Hodgkin's CeeNU ® lymphomas, primary brain tumors,small-cell lung Semustine Primary brain tumors, stomach, (methyl-CCNU)colon Streptozocin Malignant pancreatic insulinoma, (streptozotocin)malignant carcinoid Zanosar ® Triazines Dacarbazine Malignant melanoma,Hodgkin's (dimethyltriazenoimida disease, soft-tissue sarcomas,zolecarboxamide, malignant pancreatic insulinoma imidazole carboxamide)DTIC ®, DTIC-Dome ® Temozolomide Astrocytoma Temodar ®, Temodal ® MethylHydrazine Procarbazine Hodgkin's disease, glioblastoma Derivative(N-methylhydrazine, multiforme MIH) Matulane ®, Natulan ®, Indicarb ®Folic Acid Analogs Methotrexate Acute lymphocytic leukemia, Folate(amethopterin) choriocarcinoma, mycosis antimetabolites fungoides,breast, head and neck, lung, osteogenic sarcoma, glioblastomaAminopterin Leukemia Pemetrexed pleural mesothelioma, non-small Alimta ®cell lung cancer, esophageal Raltitrexed Colorectal Tomudex ® Anti-Pyrimidine Fluorouracil Breast, colon, stomach, pancreas, metabolitesAnalogs (5-fluorouracil, 5-FU, ovary, head and neck, urinary fluouracil,bladder, premalignant skin lesions fluorodeoxyuridine) (topical)Efudex ®, Carac ®, Fluoroplex ® Floxuridine (prodrug) FUDR ® Cytarabine(cytosine Acute granulocytic and acute arabinoside, ara C) lymphocyticleukemias, non- Cytosar-U ®, Tarabine Hodgkin's lymphoma PFS ®,Depocyt ® Capecitabine (prodrug) Xeloda ® Gemcitabine Pancreatic,bladder, breast, Gemzar ® oesophageal and non-small cell lung cancers,lymphomas Purine Analogs and Thioguanine Acute granulocytic, acuteRelated Inhibitors (tioguanine, lymphocytic, chronic granulocytic6-thioguanine; TG) and chronic myeloid leukemias Pentostatin Hairy cellleukemia, mycosis (2-deoxycoformycin) fungoides, chronic lymphocyticleukemia Mercaptopurine Acute lymphocytic, acute (6-mercaptopurine,granulocytic and chronic 6-MP) granulocytic leukemias, non- Purinethol ®Hodgkin's lymphoma Cladribine (2CDA) Hairy cell leukemia, BcellLeustatin ® leukemias, lymphomas Clofarabine Acute lymphoblasticleukaemia, Clolar ®, Evoltra ® acute myeloid leukaemia, juvenilemyelomonocytic leukaemia Fludarabine Hematological malignancies(fludarabine phosphate) Fludara ® Vinblastine (VLB) Hodgkin's disease,non-Hodgkin's lymphomas, breast, testis, non- small cell lung cancerVinca Alkaloids Vincristine Acute lymphocytic leukemia, Oncovin ®neuroblastoma, Wilms' tumor (nephroblastoma), rhabdomyosarcoma,Hodgkin's disease, non-Hodgkin's lymphomas, small-cell lung VindescineLeukaemia, lymphoma, melanoma, Eldisine ® breast, lung VinorelbineBreast, non-small cell lung Navelbine ® Podophyllotoxins Etoposide(etoposide Testis, small-cell lung and other Epipodo- phosphate) lung,breast, Hodgkin's disease, phyllotoxins Eposin ®, Etopophos ®,non-Hodgkin's lymphomas, acute Vepesid ®, VP-16 ® granulocytic leukemia,Kaposi's sarcoma, glioblastoma multiforme Teniposide Acute lymphocyticleukemia Vumon ®, VM-26 ® Natural Anthracycline Daunorubicin Acutegranulocytic and acute Products Antibiotics (daunomycin, lymphocyticleukemias, (Anthracyclines) rubidomycin) neuroblastoma Cerubidine ®Doxorubicin Soft-tissue, osteogenic and other (hydroxy- sarcomas;Hodgkin's disease, non- daunorubicin, Hodgkin's lymphomas, acuteadriamycin) leukemias; breast, genitourinary, Rubex ®, Doxil ® thyroid,lung, stomach, ovarian, thyroid, bladder, neuroblastoma, multiplemyeloma Epirubicin Breast, ovarian, gastric, lung; Ellence ®, lymphomasPharmorubicin ®, Ebewe ® Idarubicin (4- Acute myeloid leukemiademethoxy- daunorubicin) Zavedos ® Idamycin ® Valrubicin (N- Bladdertrifluoro-acetyl- adriamycin-14- valerate) Valstar ® AnthracenedioneMitoxantrone Acute granulocytic leukemia, breast, non-Hodgkin's lymphomaPixantrone Breast, non-Hodgkin's lymphoma Polypeptide and BleomycinTestis, head and neck, skin, peptide Antibiotics Blenoxane ® esophagus,lung and genitourinary tract; Hodgkin's disease, non- Hodgkin'slymphomas, squamous cell carcinomas Actinomycin-D Choriocarcinoma,Wilms' tumor, Dactinomycin ® rhabdomyosarcoma, testis, Kaposi's sarcomaPlicamycin Testis, malignant hypercalcemia (mithramycin) Mithracin ®Mitomycin Stomach, cervix, colon, breast, (mitomycin C) pancreas,bladder, head and neck, esophageal Enzymes L-Asparaginase Acutelymphocytic leukemia, mast Elspar ® cell tumors Biological Interferonalpha Hairy cell leukemia, Kaposi's Response (IFNα) sarcoma, melanoma,carcinoid, Modifiers Pegylated interferons renal cell, ovary, bladder,non- Multiferon ®, Hodgkin's lymphomas, mycosis Roferon ®, Pegasys ®,fungoides, multiple myeloma, IntronA ®, PegIntron ® chronic granulocyticleukemia Cytoskeletal Taxanes Taxol (paclitaxel) Breast, ovarian, lung,head and Disruptors Abraxane ® neck, Kaposi's sarcoma Docetaxel Breast,ovarian, lung, colorectal, Taxotere ® ovarian, gastric, renal, prostate,liver, head and neck, melanoma Combretastatins Combretastatin A-4Thyroid CA-4-P Platinum Cisplatin (cis-DDP, Testis, ovary, bladder, headand Coordination cisplatinum) neck, lung, thyroid, cervix, endometrium,neuroblastoma, osteogenic sarcoma, lymphoma Carboplatin Ovarian, lung,head and neck Paraplatin ® Complexes Oxaliplatin Colorectal Eloxatin ®,Oxaliplatin Medac ® Camptothecins Topotecan Ovarian, lung Hycamtin ®Irinotecan (CPT-11) Colon Camptosar Other Agents Substituted UreaHydroxyurea Chronic granulocytic leukemia, (hydroxycarbamide)polycythemia vera, essental thrombocytosis, malignant melanomaAdrenocortical Mitotane (o,p′-DDD) Adrenal cortex Lysodren ® SteroidAminoglutethimide Breast Suppressant Cytadren ® Tyrosine Kinase AxitinibBreast, renal cell carcinoma, Inhibitors pancreas Dasatinib (BMS-Chronic myelogenous leukemia, 354825) acute lymphoblastic leukemia,Sprycel ® metastatic melanoma Erlotinib (OSI-774) Non-small cell lungcancer, Tarceva ® pancreatic Gefitinib (ZD1839) Non-small cell lungcancer Iressa ® Imatinib (CGP57148B Chronic myelogenous leukemia, orSTI-571) gastrointestinal Gleevec ®, Glivec ® Lapatinib Breast(GW572016) Tykerb ®, Tyverb ® Sorafenib Renal cell carcinoma, Nexavar ®hepatocellular carcinoma Sunitinib (SU11248) Renal cell carcinoma,Sutent ® gastrointestinal, non-small cell lung cancer, breast MonoclonalReceptor tyrosine Cetuximab (anti- Colorectal, head and neck Antibodieskinases EGFR) Erbitux ® Panitumumab (anti- Colorectal EGFR) Vectibix ®Trastuzumab (anti- Breast, HER2/neu cancers HER2/neu, erbB2 receptor)Herceptin ® CD20 Rituximab Non-Hodgkin's lymphoma, B-cell Rituxan ®,leukemias MabThera ®, Reditux ® Tositumomab (anti- Follicular lymphoma,non- CD20-¹³¹I) Hodgkin's lymphoma Bexxar ® Alemtuzumab (anti- Chroniclymphocytic leukemia CD52) (CLL), T-cell lymphoma Campath ® Bevacizumab(anti- Colon, non-small cell lung cancer, VEGF) breast, renal cellcarcinoma, Avastin ® glioblastoma multiforme, hormone- refractoryprostate cancer, pancreas Gemtuzumab (anti- Acute myelogenous leukemiaCD33-calicheamicin) Mylotarg ® Adreno- Prednisone Acute and chroniclymphocytic corticosteroids leukemias, non-Hodgkin's lymphomas,Hodgkin's disease, breast, multiple myeloma ProgestinsHydroxyprogesterone Endometrium, breast caproate Medroxyprogesteroneacetate Megestrol acetate Megace ® Hormones Estrogens DiethylstilbestrolBreast, prostate and Ethinyl estradiol Antagonists Estramustine ®(mechlorethamine derivative) Antiestrogen Tamoxifen Breast Nolvadex ®,Istubal ®, Valodex ® Androgens Testosterone Breast propionateFluoxymesterone (Halotestin) Antiandrogen Flutamide (Flutamin) ProstateEulexin ® Gonadotropin- Leuprolide Prostate, breast releasing hormoneLupron ®, Lupron analog Depot ®, Viadur ®, Eligard ®, Prostap ®G2. Anti-Angiogenics

Under normal physiological conditions, humans or animals undergoangiogenesis only in very specific restricted situations. For example,angiogenesis is normally observed in wound healing, fetal and embryonicdevelopment and formation of the corpus luteum, endometrium andplacenta. Uncontrolled (persistent and/or unregulated) angiogenesis isrelated to various disease states, and occurs during tumor metastasis.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel.

The present VEGFR2-blocking, human anti-VEGF antibody may be used incombination with any one or more other anti-angiogenic therapies.Combinations with other agents that inhibit VEGF are included, such asother neutralizing antibodies (Kim et al., 1992; Presta et al., 1997;Sioussat et al., 1993; Kondo et al., 1993; Asano et al., 1995; Hurwitzet al., 2004), soluble receptor constructs (Kendall and Thomas, 1993;Aiello et al., 1995; Lin et al., 1998; Millauer et al., 1996), tyrosinekinase inhibitors (Siemeister et al., 1998), antisense strategies, RNAaptamers and ribozymes against VEGF or VEGF receptors (Saleh et al.,1996; Cheng et al., 1996; each incorporated herein by reference).Variants of VEGF with antagonistic properties may also be employed, asdescribed in WO 98/16551, specifically incorporated herein by reference.

The anti-angiogenic therapies may be based upon the provision of ananti-angiogenic agent or the inhibition of an angiogenic agentInhibition of angiogenic agents may be achieved by one or more of themethods described for inhibiting VEGF, including neutralizingantibodies, soluble receptor constructs, small molecule inhibitors,antisense, RNA aptamers and ribozymes may all be employed. For example,antibodies to angiogenin may be employed, as described in U.S. Pat. No.5,520,914, specifically incorporated herein by reference. In that FGF isconnected with angiogenesis, FGF inhibitors may also be used. Certainexamples are the compounds having N-acetylglucosamine alternating insequence with 2-O-sulfated uronic acid as their major repeating units,including glycosaminoglycans, such as archaran sulfate. Such compoundsare described in U.S. Pat. No. 6,028,061, specifically incorporatedherein by reference, and may be used in combination herewith.

Numerous tyrosine kinase inhibitors useful for the treatment ofangiogenesis, as manifest in various diseases states, are now known.These include, for example, the 4-aminopyrrolo[2,3-d]pyrimidines of U.S.Pat. No. 5,639,757, specifically incorporated herein by reference, whichmay also be used in combination with the present invention. Furtherexamples of organic molecules capable of modulating tyrosine kinasesignal transduction via the VEGFR2 receptor are the quinazolinecompounds and compositions of U.S. Pat. No. 5,792,771, which isspecifically incorporated herein by reference for the purpose ofdescribing further combinations for use with the present invention inthe treatment of angiogenic diseases.

Compounds of other chemical classes have also been shown to inhibitangiogenesis and may be used in combination with the present invention.For example, steroids such as the angiostatic 4,9(11)-steroids andC21-oxygenated steroids, as described in U.S. Pat. No. 5,972,922,specifically incorporated herein by reference, may be employed incombined therapy. U.S. Pat. Nos. 5,712,291 and 5,593,990, eachspecifically incorporated herein by reference, describe thalidomide andrelated compounds, precursors, analogs, metabolites and hydrolysisproducts, which may also be used in combination with the presentinvention to inhibit angiogenesis. The compounds in U.S. Pat. Nos.5,712,291 and 5,593,990 can be administered orally. Further exemplaryanti-angiogenic agents that are useful in connection with combinedtherapy are listed in Table D. Each of the agents listed therein areexemplary and by no means limiting.

TABLE D INHIBITORS AND NEGATIVE REGULATORS OF ANGIOGENESIS SubstancesReferences Soluble VEGFR1 Shibuya, 2006 Soluble Neuropilin-1 Gagnon etal., 2000 (NRP-1) Angiostatin O'Reilly et al., 1994 Endostatin O'Reillyet al., 1997 Angiopoietin 2 Maisonpierre et al., 1997 Calreticulin Pikeet al., 1999 Vasostatin Pike et al., 1998 Vasculostatin Kaur et al.,2005 Canstatin Kamphaus et al., 2000 Maspin Zou et al., 1994 16kDaprolactin Ferrara et al., 1991; Clapp et al., 1993; fragment D'Angelo etal., 1995; Lee et al., 1998 Laminin peptides Kleinman et al., 1993;Yamamura et al., 1993; Iwamoto et al., 1996; Tryggvason, 1993Fibronectin peptides Grant et al., 1998; Sheu et al., 1997 Tissuemetalloproteinase Sang, 1998 inhibitors (TIMP 1, 2, 3, 4) Plasminogenactivator Soff et al., 1995 inhibitors (PAI-1, -2) Tumor necrosis factorα Frater-Schroder et al., 1987 (high dose, in vitro) TGF-β1 RayChadhuryand D'Amore, 1991; Tada et al., 1994 Interferons Moore et al., 1998;Lingen et al., (IFN-α, -β, γ) 1998 ELR- CXC Chemokines: Moore et al.,1998; Hiscox and IL-12; IL-4; IL-18; Jiang, 1997; Coughlin et al., 1998;SDF-1; MIG; Tanaka et al., 1997 Platelet factor 4 (PF4); IP-10; CXCL10Thrombospondin (TSP), Good et al., 1990; Frazier, 1991; TSP-1 and TSP-2Bornstein, 1992; Tolsma et al., 1993; Sheibani and Frazier, 1995;Volpert et al., 1998 SPARC Hasselaar and Sage, 1992; Lane et al., 1992;Jendraschak and Sage, 1996 2-Methoxyoestradiol Fotsis et al., 1994Proliferin-related Jackson et al., 1994 protein Suramin Gagliardi etal., 1992; Takano et al., 1994; Waltenberger et al., 1996; Gagliardi etal., 1998; Manetti et al., 1998 Thalidomide D'Amato et al., 1994; Kenyonet al., 1997 Wells, 1998 Carboxyamidotriazole Hussain et al., 2003 (CAI)Cortisone Thorpe et al., 1993 Folkman et al., 1983 Sakamoto et al., 1986Linomide Vukanovic et al., 1993; Ziche et al., 1998; Nagler et al., 1998Fumagillin (AGM- Sipos et al., 1994; Yoshida et al., 1998 1470; TNP-470)Tamoxifen Gagliardi and Collins, 1993; Lindner and Borden, 1997; Haranet al., 1994 Korean mistletoe Yoon et al., 1995 extract (Viscum albumcoloratum) Retinoids Oikawa et al., 1989; Lingen et al., 1996; Majewskiet al. 1996 CM101 Hellerqvist et al., 1993; Quinn et al., 1995; Wamil etal., 1997; DeVore et al., 1997 Dexamethasone Hon et al., 1996; Wolff etal., 1997 Leukemia inhibitory Pepper et al., 1995 factor (LIF)

Certain preferred components for use in inhibiting angiogenesis areangiostatin, endostatin, vasculostatin, canstatin and maspin. Suchagents are described above in conjunction with the immunoconjugates ofthe present invention, but may be used in combined, but unconjugatedform.

Certain anti-angiogenic therapies have already been shown to cause tumorregressions, including the bacterial polysaccharide CM101 and theantibody LM609. CM101 is a bacterial polysaccharide that has been wellcharacterized in its ability to induce neovascular inflammation intumors. CM101 binds to and cross-links receptors expressed ondedifferentiated endothelium that stimulates the activation of thecomplement system. It also initiates a cytokine-driven inflammatoryresponse that selectively targets the tumor. It is a uniquelyantipathoangiogenic agent that downregulates the expression VEGF and itsreceptors. CM101 is currently in clinical trials as an anti-cancer drug,and can be used in combination herewith.

Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also be used incombination with the present invention. These are both angiogenesisinhibitors that associate with heparin and are found in plateletα-granules. TSP-1 is a large 450 kDa multi-domain glycoprotein that isconstituent of the extracellular matrix. TSP-1 binds to many of theproteoglycan molecules found in the extracellular matrix including,HSPGs, fibronectin, laminin, and different types of collagen. TSP-1inhibits endothelial cell migration and proliferation in vitro andangiogenesis in vivo. TSP-1 can also suppress the malignant phenotypeand tumorigenesis of transformed endothelial cells. The tumor suppressorgene p53 has been shown to directly regulate the expression of TSP-1such that, loss of p53 activity causes a dramatic reduction in TSP-1production and a concomitant increase in tumor initiated angiogenesis.

PF4 is a 70aa protein that is member of the CXC ELR-family of chemokinesthat is able to potently inhibit endothelial cell proliferation in vitroand angiogenesis in vivo. PF4 administered intratumorally or deliveredby an adenoviral vector is able to cause an inhibition of tumor growth.

Interferons and metalloproteinase inhibitors are two other classes ofnaturally occurring angiogenic inhibitors that can be combined with thepresent invention. The anti-endothelial activity of the interferons hasbeen known since the early 1980s, however, the mechanism of inhibitionis still unclear. It is known that they can inhibit endothelial cellmigration and that they do have some anti-angiogenic activity in vivothat is possibly mediated by an ability to inhibit the production ofangiogenic promoters by tumor cells. Vascular tumors in particular aresensitive to interferon, for example, proliferating hemangiomas can besuccessfully treated with IFNα.

Tissue inhibitors of metalloproteinases (TIMPs) are a family ofnaturally occurring inhibitors of matrix metalloproteases (MMPs) thatcan also inhibit angiogenesis and can be used in combined treatmentprotocols. MMPs play a key role in the angiogenic process as theydegrade the matrix through which endothelial cells and fibroblastsmigrate when extending or remodeling the vascular network. In fact, onemember of the MMPs, MMP-2, has been shown to associate with activatedendothelium through the integrin αvβ3 presumably for this purpose. Ifthis interaction is disrupted by a fragment of MMP-2, then angiogenesisis downregulated and in tumors growth is inhibited.

There are a number of pharmacological agents that inhibit angiogenesis,any one or more of which may be used in combination with the presentinvention. These include AGM-1470/TNP-470, thalidomide, andcarboxyamidotriazole (CAI). Fumagillin was found to be a potentinhibitor of angiogenesis in 1990, and since then the syntheticanalogues of fumagillin, AGM-1470 and TNP-470 have been developed. Bothof these drugs inhibit endothelial cell proliferation in vitro andangiogenesis in vivo. TNP-470 has been studied extensively in humanclinical trials with data suggesting that long-term administration isoptimal.

Thalidomide was originally used as a sedative but was found to be apotent teratogen and was discontinued. In 1994 it was found thatthalidomide is an angiogenesis inhibitor. Thalidomide is currently inclinical trials as an anti-cancer agent as well as a treatment ofvascular eye diseases.

CAI is a small molecular weight synthetic inhibitor of angiogenesis thatacts as a calcium channel blocker that prevents actin reorganization,endothelial cell migration and spreading on collagen IV. CAI inhibitsneovascularization at physiological attainable concentrations and iswell tolerated orally by cancer patients. Clinical trials with CAI haveyielded disease stabilization in 49% of cancer patients havingprogressive disease before treatment.

Cortisone in the presence of heparin or heparin fragments was shown toinhibit tumor growth in mice by blocking endothelial cell proliferation.The mechanism involved in the additive inhibitory effect of the steroidand heparin is unclear although it is thought that the heparin mayincrease the uptake of the steroid by endothelial cells. The mixture hasbeen shown to increase the dissolution of the basement membraneunderneath newly formed capillaries and this is also a possibleexplanation for the additive angiostatic effect. Heparin-cortisolconjugates also have potent angiostatic and anti-tumor effects activityin vivo.

Further specific angiogenesis inhibitors, including, but not limited to,Anti-Invasive Factor, retinoic acids and paclitaxel (U.S. Pat. No.5,716,981; incorporated herein by reference); AGM-1470 (Ingber et al.,1990; incorporated herein by reference); shark cartilage extract (U.S.Pat. No. 5,618,925; incorporated herein by reference); anionic polyamideor polyurea oligomers (U.S. Pat. No. 5,593,664; incorporated herein byreference); oxindole derivatives (U.S. Pat. No. 5,576,330; incorporatedherein by reference); estradiol derivatives (U.S. Pat. No. 5,504,074;incorporated herein by reference); and thiazolopyrimidine derivatives(U.S. Pat. No. 5,599,813; incorporated herein by reference) are alsocontemplated for use as anti-angiogenic compositions for the combineduses of the present invention.

Compositions comprising an antagonist of an α_(v)β₃ integrin may also beused to inhibit angiogenesis in combination with the present invention.As disclosed in U.S. Pat. No. 5,766,591 (incorporated herein byreference), RGD-containing polypeptides and salts thereof, includingcyclic polypeptides, are suitable examples of α_(v)β₃ integrinantagonists.

The antibody LM609 against the α_(v)β₃ integrin also induces tumorsregressions. Integrin α_(v)β₃ antagonists, such as LM609, induceapoptosis of angiogenic endothelial cells leaving the quiescent bloodvessels unaffected. LM609 or other α_(v)β₃ antagonists may also work byinhibiting the interaction of α_(v)β₃ and MMP-2, a proteolytic enzymethought to play an important role in migration of endothelial cells andfibroblasts. U.S. Pat. No. 5,753,230 is specifically incorporated hereinby reference to describe antibodies against α_(v)β₃ (vitronectinα_(v)β₃) for combined with the present invention for inhibitingangiogenesis.

Apoptosis of the angiogenic endothelium in this case may have a cascadeeffect on the rest of the vascular network Inhibiting the tumor vascularnetwork from completely responding to the tumor's signal to expand may,in fact, initiate the partial or full collapse of the network resultingin tumor cell death and loss of tumor volume. It is possible thatendostatin and angiostatin function in a similar fashion. The fact thatLM609 does not affect quiescent vessels but is able to cause tumorregressions suggests strongly that not all blood vessels in a tumor needto be targeted for treatment in order to obtain an anti-tumor effect.

Other methods of therapeutic intervention based upon altering signalingthrough the Tie2 receptor can also be used in combination with thepresent invention, such as using a soluble Tie2 receptor capable ofblocking Tie2 activation (Lin et al., 1998). Delivery of such aconstruct using recombinant adenoviral gene therapy has been shown to beeffective in treating cancer and reducing metastases (Lin et al., 1998).

G3. Apoptosis-Inducing Agents

VEGFR2-blocking, human anti-VEGF antibody therapeutic agents may also beadvantageously combined with methods to induce apoptosis. Variousapoptosis-inducing agents have been described above in connection withthe immunoconjugates of the present invention. Any suchapoptosis-inducing agent may be used in combination with the presentinvention without being linked to an antibody of the invention.

Aside from the apoptosis-inducing agents described above asimmunoconjugates, a number of oncogenes have been identified thatinhibit apoptosis, or programmed cell death. Exemplary oncogenes in thiscategory include, but are not limited to, bcr-abl, bcl-2 (distinct frombcl-1, cyclin D1; GenBank accession numbers M14745, X06487; U.S. Pat.Nos. 5,650,491; and 5,539,094; each incorporated herein by reference)and family members including Bcl-x1, Mcl-1, Bak, A1, A20. Overexpressionof bcl-2 was first discovered in T cell lymphomas. bcl-2 functions as anoncogene by binding and inactivating Bax, a protein in the apoptoticpathway Inhibition of bcl-2 function prevents inactivation of Bax, andallows the apoptotic pathway to proceed.

Inhibition of this class of oncogenes, e.g., using antisense nucleotidesequences, is contemplated for use in the present invention to giveenhancement of apoptosis (U.S. Pat. Nos. 5,650,491; 5,539,094; and5,583,034; each incorporated herein by reference).

G4. Immunotoxins and Coaguligands

The treatment methods of the invention may be used in combination withimmunotoxins and/or coaguligands in which the targeting portion thereof,e.g., antibody or ligand, is directed to a relatively specific marker ofthe tumor cells, tumor vasculature or tumor stroma. In common with thechemotherapeutic and anti-angiogenic agents discussed above, thecombined use of targeted toxins or coagulants will generally result inadditive, markedly greater than additive or even synergistic anti-tumorresults.

Generally speaking, antibodies or ligands for use in these additionalaspects of the invention will preferably recognize accessible tumorantigens that are preferentially, or specifically, expressed in thetumor site. The antibodies or ligands will also preferably exhibitproperties of high affinity; and the antibodies, ligands or conjugatesthereof, will not exert significant in vivo side effects againstlife-sustaining normal tissues, such as one or more tissues selectedfrom heart, kidney, brain, liver, bone marrow, colon, breast, prostate,thyroid, gall bladder, lung, adrenals, muscle, nerve fibers, pancreas,skin, or other life-sustaining organ or tissue in the human body. Theterm “significant side effects”, as used herein, refers to an antibody,ligand or antibody conjugate, that, when administered in vivo, willproduce only negligible or clinically manageable side effects, such asthose normally encountered during chemotherapy.

At least one binding region of these second anti-cancer agents employedin combination with the invention will be a component that is capable ofdelivering a toxin or coagulation factor to the tumor region, i.e.,capable of localizing within a tumor site. Such targeting agents may bedirected against a component of a tumor cell, tumor vasculature or tumorstroma. The targeting agents will generally bind to a surface-expressed,surface-accessible or surface-localized component of a tumor cell, tumorvasculature or tumor stroma. However, once tumor vasculature and tumorcell destruction begins, internal components will be released, allowingadditional targeting of virtually any tumor component.

Many tumor cell antigens have been described, any one which could beemployed as a target in connection with the combined aspects of thepresent invention. Appropriate tumor cell antigens for additionalimmunotoxin and coaguligand targeting include those recognized by theantibodies B3 (U.S. Pat. No. 5,242,813); incorporated herein byreference; ATCC HB 10573); KSI/4 (U.S. Pat. No. 4,975,369); incorporatedherein by reference; obtained from a cell comprising the vectors NRRLB-18356 and/or NRRL B-18357); 260F9 (ATCC HB 8488); and D612 (U.S. Pat.No. 5,183,756); incorporated herein by reference; ATCC HB 9796). One mayalso consult the ATCC Catalogue of any subsequent year to identify otherappropriate cell lines producing anti-tumor cell antibodies.

For tumor vasculature targeting, the targeting antibody or ligand willoften bind to a marker expressed by, adsorbed to, induced on orotherwise localized to the intratumoral blood vessels of a vascularizedtumor. Appropriate expressed target molecules include, for example,endoglin, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA (Liu et al.,1997), a TIE, a ligand reactive with LAM-1, a VEGF/VPF receptor, an FGFreceptor, α_(v)β₃ integrin, pleiotropin and endosialin. Suitableadsorbed targets are those such as VEGF, FGF, TGFβ, HGF, PF4, PDGF,TIMP, a ligand that binds to a TIE and tumor-associated fibronectinisoforms. Antigens naturally and artificially inducible by cytokines andcoagulants may also be targeted, such as ELAM-1, VCAM-1, ICAM-1, aligand reactive with LAM-1, endoglin, and even MHC Class II(cytokine-inducible, e.g., by IL-1, TNF-α, IFN-γ, IL-4 and/or TNF-β);and E-selectin, P-selectin, PDGF and ICAM-1 (coagulant-inducible e.g.,by thrombin, Factor IX/IXa, Factor X/Xa and/or plasmin).

The following patents are specifically incorporated herein by referencefor the purposes of even further supplementing the present teachingsregarding the preparation and use of immunotoxins directed againstexpressed, adsorbed, induced or localized markers of tumor vasculature:U.S. Pat. Nos. 6,093,399; 5,855,866; 5,965,132; 6,051,230; 6,004,555;5,877,289; 6,004,554; 5,776,427; 5,863,538; 5,660,827 and 6,036,955.

Further tumor vasculature targeting compositions and methods includethose targeting aminophospholipids, such as phosphatidylserine andphosphatidylethanolamine, recently discovered to be accessible, specificmarkers of tumor blood vessels. Administration of anti-aminophospholipidantibodies alone is sufficient to induce thrombosis and tumorregression. The present invention can thus be effectively combined withunconjugated, anti-phosphatidylserine and/or phosphatidylethanolamineantibodies; or immunoconjugates of such antibodies can be used.

The following patents are specifically incorporated herein by referencefor the purposes of even further supplementing the present teachingsregarding the preparation and use of anti-aminophospholipid antibodiesand immunotoxins: U.S. Pat. Nos. 6,406,693; 6,312,694; 6,783,760;6,818,213; and 7,067,109. U.S. Pat. Nos. 6,312,694; 6,783,760;6,818,213; and 7,067,109 are further incorporated herein by referencefor the purposes of further supplementing the present teachingsregarding the use of aminophospholipid binding protein conjugates, suchas annexin conjugates, for use in delivering toxins and coagulants totumor blood vessels and for inducing thrombosis and tumor regression.

Suitable tumor stromal targets include components of the tumorextracellular matrix or stroma, or components those bound therein;including basement membrane markers, type IV collagen, laminin, heparansulfate, proteoglycan, fibronectins, activated platelets, FIBS andtenascin. A preferred target for such uses is RIBS.

The following patents are specifically incorporated herein by referencefor the purposes of even further supplementing the present teachingsregarding the preparation and use of tumor stromal targeting agents:U.S. Pat. Nos. 6,093,399; 6,004,555; 5,877,289; and 6,036,955.

The second anti-cancer therapeutics may be operatively attached to anyof the cytotoxic or otherwise anti-cellular agents described herein foruse in the VEGFR2-blocking, anti-VEGF antibody or the VEGFR2-blocking,anti-VEGF antibody-based immunotoxins. However, suitable anti-cellularagents also include radioisotopes. Toxin moieties will be preferred,such as ricin A chain and deglycosylated A chain (dgA).

The second, targeted agent for optional use with the invention maycomprise a targeted component that is capable of promoting coagulation,i.e., a coaguligand. Here, the targeting antibody or ligand may bedirectly or indirectly, e.g., via another antibody, linked to any factorthat directly or indirectly stimulates coagulation, including any ofthose described herein for use in the VEGFR2-blocking, anti-VEGFantibody or VEGFR2-blocking, anti-VEGF antibody-based coaguligands.Preferred coagulation factors for such uses are Tissue Factor (TF) andTF derivatives, such as truncated TF (tTF), dimeric and multimeric TF,and mutant TF deficient in the ability to activate Factor VII.

Effective doses of immunotoxins and coaguligands for combined use in thetreatment of cancer will be between about 0.1 mg/kg and about 2 mg/kg,and preferably, of between about 0.8 mg/kg and about 1.2 mg/kg, whenadministered via the IV route at a frequency of about 1 time per week.Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The physician responsible foradministration will determine the appropriate dose for the individualsubject.

G5. TLR Agonists

It has now been established that signaling via Toll-Like Receptors(TLRs) contributes to the effects of known anti-cancer agents, includingattenuated S. choleraesuis, BCG and taxol, which each activate TLR4.Indeed, TLR4 signaling contributes to the anti-cancer effects ofchemotherapy and radiotherapy (Apetoh et al., 2007). As well as betterunderstanding the mechanisms of action of certain known anti-canceragents, recognizing the importance of TLR signaling has also promptedthe development of new cancer therapeutics that function by activatingTLRs.

Therefore, the VEGFR2-blocking, human anti-VEGF antibodies of thepresent invention may be used in cancer treatment in combination withone or more agents that stimulate signaling via a TLR, i.e., with one ormore TLR agonists. At least a first TLR agonist may also be operativelyattached to a human antibody of the invention to create a therapeuticconjugate, as described herein in the immunoconjugate section. Any oneor more of the following or other TLR agonists may be used in thepresent combination cancer treatments.

Suitable TLR agonists include agonists of any one or more of TLR1 toTLR11, preferably TLR1, TLR2, TLR4, TLR7, TLR8 or TLR9, and mostpreferably TLR4, TLR7, TLR8 or TLR9. TLR1/TLR2 agonists includelipoproteins, e.g., OspA, and triacylated lipopeptides, and TLR2agonists include bacterial lipoproteins, LAM, MALP-2, GPI, glycolipidsand porins.

Particular examples of TLR4 agonists include the agonistic anti-TLR4antibody termed 5D24.D4 (Cohen et al., 2003), LPS, lipid A andderivatives thereof, of which monophosphoryl lipid A (MPL) and MPLanalogues are currently preferred. MPL analogues known as AGPs may beused as synthetic TLR4 agonists in combination with the presentinvention (Alderson et al., 2006). Agonists stimulating signaling viaTLR4 and CD14 also include LPS, lipid A, MPL and MPL analogues, as wellas taxol, paclitaxel, flavolipin and GIPLs. The TLR4 agonists OK-432 andOK-PSA have been used to treat cervical cancer and non-small-cell lungcarcinoma.

TLR7 agonists include imiquimod, resiquimod and isatoribine (Finberg etal., 2005; Horsmans et al., 2005), and imiquimod is approved for use totreat basal cell carcinoma. Other TLR7 agonists include gardiquimod,loxoribine and bropirimine. Resiquimod is also a TLR8 agonist. TLR9agonists, such as CpG, have been used to treat non-small-cell lungcarcinoma, non-Hodgkin's lymphoma, renal cell carcinoma and colorectalcancer.

G6. ADEPT and Prodrug Therapy

The VEGFR2-blocking, human anti-VEGF antibodies of the present inventionmay be used in conjunction with prodrugs, wherein the VEGFR2-blocking,human anti-VEGF antibody is operatively associated with aprodrug-activating component, such as a prodrug-activating enzyme, whichconverts a prodrug to the more active form only upon contact with theantibody. This technology is generally termed “ADEPT”, and is describedin, e.g., WO 95/13095; WO 97/26918, WO 97/24143, and U.S. Pat. Nos.4,975,278 and 5,658,568, each specifically incorporated herein byreference.

The term “prodrug”, as used herein, refers to a precursor or derivativeform of a biologically or pharmaceutically active substance that exertsreduced cytotoxic or otherwise anticellular effects on targets cells,including tumor vascular endothelial cells, in comparison to the parentdrug upon which it is based. Preferably, the prodrug or precursor formexerts significantly reduced, or more preferably, negligible, cytotoxicor anticellular effects in comparison to the “native” or parent form.“Prodrugs” are capable of being activated or converted to yield the moreactive, parent form of the drug.

The technical capability to make and use prodrugs exists within theskill of the ordinary artisan. Willman et al. (1988) and Stella andHimmelstein (1985) are each specifically incorporated herein byreference for purposes of further supplementing the description andteaching concerning how to make and use various prodrugs. Exemplaryprodrug constructs that may be used in the context of the presentinvention include, but are not limited to, phosphate-containing prodrugs(U.S. Pat. No. 4,975,278), thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-based prodrugs (U.S. Pat. Nos.5,660,829; 5,587,161; 5,405,990; WO 97/07118), D-amino acid-modifiedprodrugs, glycosylated prodrugs (U.S. Pat. Nos. 5,561,119; 5,646,298;4,904,768, 5,041,424), β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs (U.S. Pat. No.4,975,278), optionally substituted phenylacetamide-containing prodrugs,and even 5-fluorocytosine (U.S. Pat. No. 4,975,278) and 5-fluorouridineprodrugs and the like, wherein each of the patents are specificallyincorporated herein by reference.

The type of therapeutic agent or cytotoxic drug that can be used inprodrug form is virtually limitless. The more cytotoxic agents will bepreferred for such a form of delivery, over, e.g., the delivery ofcoagulants, which are less preferred for use as prodrugs. All that isrequired in forming the prodrug is to design the construct so that theprodrug is substantially inactive and the “released” or activated drughas substantial, or at least sufficient, activity for the intendedpurpose.

Various improvements on the original prodrugs are also known andcontemplated for use herewith, as disclosed in WO 95/03830; EP 751,144(anthracyclines); WO 97/07097 (cyclopropylindoles); and WO 96/20169. Forexample, prodrugs with reduced Km are described in U.S. Pat. No.5,621,002, specifically incorporated herein by reference, which may beused in the context of the present invention. Prodrug therapy that beconducted intracellularly is also known, as exemplified by WO 96/03151,specifically incorporated herein by reference, and can be practicedherewith.

For use in ADEPT, the agent that activates or converts the prodrug intothe more active drug is operatively attached to the VEGFR2-blocking,human anti-VEGF antibody. The VEGFR2-blocking, human anti-VEGF antibodythus localizes the prodrug converting capability within the angiogenicsite, preferably, within the tumor vasculature and stroma, so thatactive drug is only produced in such regions and not in circulation orin healthy tissues.

Enzymes that may be attached to VEGFR2-blocking, human anti-VEGFantibodies to function in prodrug activation include, but are notlimited to, alkaline phosphatase for use in combination withphosphate-containing prodrugs (U.S. Pat. No. 4,975,278); arylsulfatasefor use in combination with sulfate-containing prodrugs (U.S. Pat. No.5,270,196); peptidases and proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidase (U.S. Pat. Nos. 5,660,829;5,587,161; 5,405,990) and cathepsins (including cathepsin B and L), foruse in combination with peptide-based prodrugs;D-alanylcarboxypeptidases for use in combination with D-aminoacid-modified prodrugs; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase for use in combination withglycosylated prodrugs (U.S. Pat. Nos. 5,561,119; 5,646,298); β-lactamasefor use in combination with β-lactam-containing prodrugs; penicillinamidases, such as penicillin V amidase (U.S. Pat. No. 4,975,278) orpenicillin G amidase, for use in combination with drugs derivatized attheir amino nitrogens with phenoxyacetamide or phenylacetamide groups;and cytosine deaminase (U.S. Pat. Nos. 5,338,678; 5,545,548) for use incombination with 5-fluorocytosine-based prodrugs (U.S. Pat. No.4,975,278), wherein each of the patents are specifically incorporatedherein by reference.

Antibodies with enzymatic activity, known as catalytic antibodies or“abzymes”, can also be employed to convert prodrugs into active drugs.Abzymes based upon VEGFR2-blocking, human anti-VEGF antibodies thus formanother aspect of the present invention. The technical capacity to makeabzymes also exists within one of ordinary skill in the art, asexemplified by Massey (1987), specifically incorporated herein byreference for purposes of supplementing the abzyme teaching. Catalyticantibodies capable of catalyzing the breakdown of a prodrug at thecarbamate position, such as a nitrogen mustard aryl carbamate, arefurther contemplated, as described in EP 745,673, specificallyincorporated herein by reference.

G7. Ocular Combinations

The VEGFR2-blocking, human anti-VEGF antibodies of the invention may beused in combination with other therapies to treat ocular diseases andangiogenic ocular diseases, including diabetic retinopathy, maculardegeneration, age-related macular degeneration, neovascular glaucoma andthe other ocular diseases described above. The antibodies may becombined with any other methods generally employed in the treatment ofocular diseases, including surgery.

As to combinations with other therapeutic agents, the VEGFR2-blocking,human anti-VEGF antibodies may be administered before, after or atsubstantially the same time as the other therapeutic agent.Substantially simultaneous administration may be achieved from a singlecomposition, or from two distinct compositions.

As to choroidal neovascularization, such as that associated with maculardegeneration, age-related macular degeneration (AMD) and other ocularindications, certain preferred combinations of the invention are thoseusing a second agent that blocks, inhibits, reduces, down-regulates orantagonizes SPARC (secreted protein, acidic and rich in cysteine)(Nozaki et al., 2006; U.S. 2006/0135423). As the antibodies of theinvention already block VEGFR2 activation, but not VEGFR1 activation,their combination with one or more agents that block SPARC would form aparticularly effective method to further reduce VEGF-inducedangiogenesis in the eye.

SPARC inhibitors or antagonists include, for example, those of the samemolecular types as have been successfully developed against VEGF itself.Exemplary SPARC inhibitors thus include inhibitory anti-SPARC antibodiesand antigen-binding fragments thereof (e.g., Sweetwyne et al., 2004);antisense strategies, such as RNA aptamers and RNA/DNA aptamers,silencing RNAs (siRNAs or RNAi) that silence or interfere with SPARCexpression; ribozymes; and other protein, peptide and small moleculeinhibitors. Many such SPARC inhibitors, including polyclonal andmonoclonal antibodies and siRNAs, are available commercially, e.g., fromSigma/Aldrich, Santa Cruz Biotechnology, Inc., R&D Systems. Any one ormore SPARC inhibitors may thus be used in conjunction with the presentinvention to additionally block, inhibit, reduce, down-regulate orantagonize SPARC levels or activity, either at the DNA, RNA and/orprotein levels.

H. Diagnostics and Imaging

The present invention further provides in vitro and in vivo diagnosticand imaging methods. Such methods are applicable for use in generatingdiagnostic, prognostic or imaging information for any angiogenicdisease, as exemplified by arthritis, psoriasis and solid tumors, butincluding all the angiogenic diseases disclosed herein. Outside thefield of tumor diagnostics and imaging, these aspects of the inventionare most preferred for use in in vitro diagnostic tests, preferablyeither where samples can be obtained non-invasively and tested in highthroughput assays and/or where the clinical diagnosis in ambiguous andconfirmation is desired.

H1. Immunodetection Methods and Kits

In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifying orotherwise generally detecting VEGF and for diagnosing angiogenicdiseases. The VEGFR2-blocking, human anti-VEGF antibodies of the presentinvention may be employed to detect VEGF in vivo (see below), inisolated issue samples, biopsies or swabs and/or in homogenized tissuesamples. Such immunodetection methods have evident diagnostic utility,but also have applications to non-clinical samples, such as in thetitering of antigen samples, and the like.

The steps of various useful immunodetection methods have been describedin the scientific literature, such as, e.g., Nakamura et al. (1986,incorporated herein by reference). In general, the immunobinding methodsinclude obtaining a sample suspected of containing VEGF and contactingthe sample with VEGFR2-blocking, human anti-VEGF antibodies underconditions effective to allow the formation of immunocomplexes. In suchmethods, the antibody may be linked to a solid support, such as in theform of a column matrix, and the sample suspected of containing VEGFwill be applied to the immobilized antibody.

More preferably, the immunobinding methods include methods for detectingor quantifying the amount of VEGF in a sample, which methods require thedetection or quantification of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containingVEGF and contact the sample with an antibody in accordance herewith andthen detect or quantify the amount of immune complexes formed under thespecific conditions.

The biological sample analyzed may be any sample that is suspected ofcontaining VEGF, generally from an animal or patient suspected of havingan angiogenic disease. The samples may be a tissue section or specimen,a biopsy, a swab or smear test sample, a homogenized tissue extract orseparated or purified forms of such.

Contacting the chosen biological sample with the antibody underconditions effective and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding an antibody composition to the sample andincubating the mixture for a period of time lone enough for theantibodies to form immune complexes with, i.e., to bind to, any VEGFpresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those antibodies specifically bound within the primary immunecomplexes to be detected.

The detection of immunocomplex formation is well known in the art andmay be achieved through the application of numerous approaches. Thesemethods are generally based upon the detection of a label or marker,such as any radioactive, fluorescent, biological or enzymatic tags orlabels known in the art. U.S. patents concerning the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241, each incorporated herein byreference. The use of enzymes that generate a colored product uponcontact with a chromogenic substrate are generally preferred. Secondarybinding ligand, such as a second antibody or a biotin/avidin ligandbinding arrangement, may also be used, as is known in the art.

The VEGFR2-blocking, human anti-VEGF antibodies employed in thedetection may themselves be linked to a detectable label, wherein onewould then simply detect this label, thereby allowing the amount of theprimary immune complexes in the composition to be determined

Preferably, the primary immune complexes are detected by means of asecond binding ligand that has binding affinity for the antibodies ofthe invention. In such cases, the second binding ligand may be linked toa detectable label. The second binding ligand is itself often anantibody, and may thus be termed a “secondary” antibody. The primaryimmune complexes are contacted with the labeled, secondary bindingligand, or antibody, under conditions effective and for a period of timesufficient to allow the formation of secondary immune complexes. Thesecondary immune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the first antibody is used to form secondaryimmune complexes, as described above. After washing, the secondaryimmune complexes are contacted with a third binding ligand or antibodythat has binding affinity for the second antibody, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (tertiary immune complexes). The thirdligand or antibody is linked to a detectable label, allowing detectionof the tertiary immune complexes thus formed. This system may providefor signal amplification if desired.

In the clinical diagnosis or monitoring of patients with an angiogenicdisease, the detection of VEGF, or an increase in the levels of VEGF, incomparison to the levels in a corresponding biological sample from anormal subject is indicative of a patient with an angiogenic disease.

However, as is known to those of skill in the art, such a clinicaldiagnosis would not likely be made on the basis of this method inisolation. Those of skill in the art are very familiar withdifferentiating between significant expression of a biomarker, whichrepresents a positive identification, and low level or backgroundexpression of a biomarker. Indeed, background expression levels areoften used to form a “cut-off” above which increased staining will bescored as significant or positive.

H2. Imaging

These aspects of the invention are preferred for use in tumor imagingmethods and combined tumor treatment and imaging methods.VEGFR2-blocking, human anti-VEGF antibodies that are linked to one ormore detectable agents are envisioned for use in imaging per se, or forpre-imaging the tumor to form a reliable image prior to treatment. Suchcompositions and methods can also be applied to the imaging anddiagnosis of any other angiogenic disease or condition, particularlynon-malignant tumors, atherosclerosis and conditions in which aninternal image is desired for diagnostic or prognostic purposes or todesign treatment.

VEGFR2-blocking, human anti-VEGF antibody imaging antibodies willgenerally comprise a VEGFR2-blocking, human anti-VEGF antibodyoperatively attached, or conjugated to, a detectable label. “Detectablelabels” are compounds or elements that can be detected due to theirspecific functional properties, or chemical characteristics, the use ofwhich allows the component to which they are attached to be detected,and further quantified if desired. In antibody conjugates for in vivodiagnostic protocols or “imaging methods” labels are required that canbe detected using non-invasive methods.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies and binding ligands (see, e.g., U.S. Pat.Nos. 5,021,236 and 4,472,509, both incorporated herein by reference).Certain attachment methods involve the use of a metal chelate complexemploying, for example, an organic chelating agent such a DTPA attachedto the antibody (U.S. Pat. No. 4,472,509). Monoclonal antibodies mayalso be reacted with an enzyme in the presence of a coupling agent suchas glutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate.

An example of detectable labels are the paramagnetic ions. In this case,suitable ions include chromium (III), manganese (II), iron (III), iron(II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium(III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), with gadolinium beingparticularly preferred.

Ions useful in other contexts, such as X-ray imaging, include but arenot limited to lanthanum (III), gold (III), lead (II), and especiallybismuth (III). Fluorescent labels include rhodamine, fluorescein andrenographin. Rhodamine and fluorescein are often linked via anisothiocyanate intermediate.

In the case of radioactive isotopes for diagnostic applications,suitable examples include ¹⁴-carbon, ⁵¹chromium, ³⁶-chlorine, ⁵⁷cobalt,⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹²⁵,iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸,⁷⁵selenium, ³⁵sulphur, technetium^(99m) and yttrium⁹⁰. ¹²⁵I is oftenbeing preferred for use in certain embodiments, and technicium^(99m) andindium¹¹¹ are also often preferred due to their low energy andsuitability for long range detection.

Radioactively labeled VEGFR2-blocking, human anti-VEGF antibodyantibodies for use in the present invention may be produced according towell-known methods in the art. For instance, intermediary functionalgroups that are often used to bind radioisotopic metallic ions toantibodies are diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetracetic acid (EDTA).

Monoclonal antibodies can also be iodinated by contact with sodium orpotassium iodide and a chemical oxidizing agent such as sodiumhypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.Antibodies according to the invention may be labeled with technetium-⁹⁹mby ligand exchange process, for example, by reducing pertechnate withstannous solution, chelating the reduced technetium onto a Sephadexcolumn and applying the antibody to this column; or by direct labelingtechniques, e.g., by incubating pertechnate, a reducing agent such asSNCl₂, a buffer solution such as sodium-potassium phthalate solution,and the antibody.

Any of the foregoing type of detectably labeled VEGFR2-blocking, humananti-VEGF antibodies may be used in the imaging or combined imaging andtreatment aspects of the present invention. They are equally suitablefor use in in vitro diagnostics. Dosages for in vivo imaging embodimentsare generally less than for therapy, but are also dependent upon the ageand weight of a patient. One time doses should be sufficient.

The in vivo diagnostic or imaging methods generally compriseadministering to a patient a diagnostically effective amount of aVEGFR2-blocking, human anti-VEGF antibody that is conjugated to a markerthat is detectable by non-invasive methods. The antibody-markerconjugate is allowed sufficient time to localize and bind to VEGF withinthe tumor. The patient is then exposed to a detection device to identifythe detectable marker, thus forming an image of the tumor.

H3. Diagnostic Kits

In still further embodiments, the present invention provides diagnostickits, including both immunodetection and imaging kits, for use with theimmunodetection and imaging methods described above. Accordingly, theVEGFR2-blocking, human anti-VEGF antibodies are provided in the kit,generally comprised within a suitable container.

For immunodetection, the antibodies may be bound to a solid support,such as a well of a microtitre plate, although antibody solutions orpowders for reconstitution are preferred. The immunodetection kitspreferably comprise at least a first immunodetection reagent. Theimmunodetection reagents of the kit may take any one of a variety offorms, including those detectable labels that are associated with orlinked to the given antibody. Detectable labels that are associated withor attached to a secondary binding ligand are also contemplated.Exemplary secondary ligands are those secondary antibodies that havebinding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted above, a number ofexemplary labels are known in the art and all such labels may beemployed in connection with the present invention. These kits maycontain antibody-label conjugates either in fully conjugated form, inthe form of intermediates, or as separate moieties to be conjugated bythe user of the kit.

The imaging kits will preferably comprise a VEGFR2-blocking, humananti-VEGF antibody that is already attached to an in vivo detectablelabel. However, the label and attachment means could be separatelysupplied.

Either kit may further comprise control agents, such as suitablyaliquoted compositions of VEGF, whether labeled or unlabeled, as may beused to prepare a standard curve for a detection assay. The componentsof the kits may be packaged either in aqueous media or in lyophilizedform.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antibody or antigen may be placed, and preferably, suitablyaliquoted. Where a second or third binding ligand or additionalcomponent is provided, the kit will also generally contain a second,third or other additional container into which this ligand or componentmay be placed. The kits may also include other diagnostic reagents foruse in the diagnosis of any one or more angiogenic diseases. Preferably,second diagnostics not based upon VEGF binding will be used.

The kit of the present invention will also typically include a means forcontaining the antibody, and any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionor blow-molded plastic containers into which the desired vials areretained.

TABLE 1 SEQ ID  NO: Description Sequence Clone EJ173-112-Cl1 (r84 scFv) 1 VH domain (nt) CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACA AGGGCTTGAGTGGATGGGAGGTTTTGATCCTGAAGATGGTGAAACAATCTACGCACAGAAGTTC CAGGGCAGAGTCACCATGACCGAGGACACATCTACAGACACAGCCTACATGGAGCTGAGCAGCC TGAGATCTGAGGACACGGCCGTGTATTACTGTGCAACAGGACGTTCTATGGTTCGGGGAGTCAT TATACCTTTTAACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA See Figure 1  2 VL domain (nt)GACATCCGGATGACCCAGTCTCCATCCTCCCT GTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTAT TTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTT TGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT CAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGCTC ACTTTCGGCGGAGGGACCAAGGTGGAGATCAA ASee Figure 1  3 VH domain (aa) QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGFDPEDGETIYAQKF QGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGRSMVRGVIIPFNGMDVWGQGTTVTVSS See Figure 1  4 VL domain (aa)DIRMTQSPSSLSASVGDRVTITCRASQSISSY LNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPL TFGGGTKVEIK See Figure 1  5 Heavy CDR1SYAIS  6 Heavy CDR2 GFDPEDGETIYAQKFQG  7 Heavy CDR3 GRSMVRGVIIPFNGMDV  8Light CDR1 RASQSISSYLN  9 Light CDR2 AASSLQS 10 Light CDR3 QQSYSTPLT 11Heavy FR1 QVQLVQSGAEVKKPGASVKVSCKASGGTFS 12 Heavy FR2 WVRQAPGQGLEWMG 13Heavy FR3 RVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT 14 Heavy FR4 WGQGTTVTVSS 15Light FR1 DIRMTQSPSSLSASVGDRVTITC 16 Light FR2 WYQQKPGKAPKLLIY 17Light FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 18 Light FR4 FGGGTKVEIK 19Linker KLSGSASAPKLEEGEFSEARV 20 Whole scFv clone See Figure 1 (nt) 21Whole scFv clone See Figure 1 (aa) r84 Full length IgG 22IgG heavy chain See Example 6 (nt) 23 IgG light chain (nt) See Example 624 IgG heavy chain See Example 6 (aa) 25 IgG light chain See Example 6(aa) 26 IgG VH domain See Example 6 (nt) 27 IgG VL domain See Example 6(nt)

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES Example 1 Antibody Selection

VEGF is a key regulator of physiological angiogenesis duringembryogenesis, skeletal growth, and reproductive functions. VEGFsignaling through interaction with the tyrosine kinase receptor VEGFR2is also important in pathological angiogenesis, including thatassociated with tumor growth. Given the need for therapeutic specifichuman antibodies that block angiogenesis, human antibodies have beenidentified that are reactive against an epitope on VEGF thatspecifically and substantially blocks its interaction with VEGFR2(KDR/Flk-1), but does not substantially block its interaction withVEGFR1 (Flt-1).

Single chain forms of antibodies were cloned in the pHOG21plasmid(Kipriyanov et al., 1996; 1997) (FIG. 9A and FIG. 9B) (at the NcoI andNot I restriction sites), which contains c-myc and 6×His tag epitopes.E. coli cells, XL-1 blue, were transformed, selected on ampicillinplates and the scFv was expressed upon IPTG induction. Purified scFvwere tested by ELISA for selective biological activity against VEGF. Theselective biological activity was further confirmed by competitive ELISAassays using the murine antibody 2C3, which specifically blocks VEGF andVEGFR2 interaction and not VEGF and VEGFR1 interaction (Brekken et al.,1998; 2000). Also Biacore showed the binding of scFv antibodies toimmobilized VEGF-A. Binding to murine VEGF as well as human VEGF wasalso assessed.

A. Sequencing

The nucleotide sequences of the heavy and light chains of one preferredantibody-producing clone is shown. The antibody is designated asEJ173/112-C11 (r84/PGN311) and has been produced in both a scFv form(Example 1 and FIG. 1) and a full length IgG form (Example 6). Thenucleotide sequence and amino acid sequence of the light and heavychains of a single chain form of EJ173/112-C11 (r84/PGN311) are shown inFIG. 1 and Table 1. The nucleotide and amino acid sequence of the lightand heavy chains of a full length IgG form of r84/PGN311 are shown inExample 6. The CDR and framework regions of the light and heavy chainsof EJ173/112-C11 (r84/PGN311) are shown in Table 1.

Example 2 EJ173/112-C11 (r84/PGN311) Binds to VEGF with High Affinity

To confirm the specificity of the antibody, binding of the scFv form ofEJ173/112-C11 (r84/PGN311) was tested by ELISA against plated humanVEGF-A (obtained from Dr. Rolf A. Brekken, UT Southwestern MedicalCenter, Dallas, Tex.). Briefly, 2 μg/ml VEGF-A was plated on apolystyrene plate. Next, 20 μg/ml purified EJ173/112-C11 (r84/PGN311)scFv was added to the first well, and titrated with 3-fold dilutions.Bound scFv was detected with an anti-c-myc tag mouse monoclonal antibody(Invitrogen) and HRP-conjugated secondary rabbit anti-mouse antibody.

ELISA results showed that EJ173/112-C11 (r84/PGN311) scFv (FIG. 2) boundto VEGF and, importantly, had an increased binding signal, and henceincreased affinity, compared to its mother clone. The murine B9 antibodyis used as a positive control and is a murine scFv antibody againsthuman VEGF-A (obtained from Dr. Philip E. Thorpe, UT SouthwesternMedical Center, Dallas, Tex.).

EJ173/112-C11 (r84/PGN311) showed further beneficial features over themother clone. It was shown that EJ173/112-C11 (r84/PGN311) has a higherstability in serum and a reduced tendency to form aggregates in scFvformat compared to the mother clone (data not shown).

Shuffling the Variable Region Heavy Chain of EJ1731112-C11 (r84/PGN311)with Seven Different Heavy Chains from Other Anti-VEGF Antibodies

The variable region light chain of EJ173/112-C11 (r84/PGN311) wascombined with seven different variable region heavy chains derived fromother anti□VEGF antibody clones distinct from r84/PGN311 to confirm theimportance of the light chain variable region of r84/PGN311 inmaintaining the VEGF binding property. The resulting clones wereexpressed and purified via their His tag on NiNTA columns. Afterpurification, concentration was determined, and an ELISA against platedhuman VEGF-A was run. 20 μg/ml of purified scFv was added and bound scFvwas detected with an anti-c-myc tag mouse monoclonal antibody(Invitrogen) and HRP-conjugated secondary rabbit anti-mouse antibody.

It was shown that three out of the seven combinations of variable regionlight chain of EJ173/112-C11 (r84/PGN311) with variable region heavychains derived from other□ anti-VEGF antibody clones showed significantbinding to VEGF in this ELISA. This is a very reasonable proportion anddemonstrates that the light chain variable region of r84/PGN311 isimportant for maintaining the binding to VEGF and also that other heavychain variable regions which can be combined with this light chainvariable region to give rise to antibodies which bind to VEGF can bereadily identified.

Example 3 EJ173/112-C11 (r84/PGN311) Competes with Murine 2C3

To further demonstrate the specificity of the antibody, binding ofEJ173/112-C11 (r84/PGN311) scFv in the presence of two concentrations of2C3 was tested in an ELISA against plated VEGF-A. Briefly, 2 μg/mlVEGF-A was plated on a polystyrene plate. Next, 1 μg/ml purifiedEJ173/112-C11 (r84/PGN311) scFv, the mother clone or murine B9 scFv(FIG. 3) was added to six parallel wells, of which two contained 0.1 μgand two contained 1 μg murine 2C3 IgG, resulting in a finalconcentration of 1 and 10 μg/ml, respectively, of 2C3 IgG. Remainingbound scFv was detected with an HRP-conjugated anti-c-myc tag mousemonoclonal antibody (Invitrogen).

The binding of EJ173/112-C11 (r84/PGN311) scFv to VEGF was reduced bycompetition with increasing concentrations of 2C3 IgG. These resultstherefore show that EJ173/112-C11 (r84/PGN311) effectively competes withthe 2C3 antibody for binding to VEGF, indicating that EJ173/112-C11(r84/PGN311) binds to substantially the same epitope as 2C3.

Example 4 EJ173/112-C11 (r84/PGN311) Binds to Human and Mouse VEGF

The binding of EJ173/112-C11 (r84/PGN311) scFv to human and murine VEGFwas determined. 1 μg/ml of murine VEGF (R&D Systems 493-MV-005/CF,carrier-free murine VEGF164) and human VEGF was plated on polystyreneimmunoplates. 10 μg/ml of purified scFv was added and detected with ananti-c-myc tag mouse monoclonal antibody (Invitrogen) and HRP-conjugatedsecondary rabbit anti-mouse antibody.

The results showed that EJ173/112-C11 (r84/PGN311) scFv (FIG. 4) bindsto both murine VEGF and human VEGF.

In addition, a Biacore T100 was used to assess the binding affinity ofthe scFv forms of r84 and its mother clone to mouse VEGF. To this end1000 RU of recombinant Mouse VEGF₁₆₄ (493-MV/CF, R&D Systems) wasimmobilised on a CM5 chip (Biacore), and a dilution series (100 nM and2-fold dilutions) of monomeric scFv was flown over at a flow rate of 50μl/min. The binding affinity expressed as the K_(D) was calculated bythe 1:1 Fitting model using software belonging to the Biacore T100machine. The K_(D) values were calculated as 1.0×10⁻⁸ M forEJ173/112-C11 (r84/PGN311) and 4.0×10⁻⁸ M for the mother clone.r84/PGN311 thus shows a higher binding affinity than the mother clonefor murine VEGF.

These results indicate that this selected antibody (r84/PGN311) issuitable for use both in pre-clinical studies in mice and for use inhumans.

As detailed below in Example 6, fully human and murine chimeric IgGforms of the r84 antibody have been generated. ELISA binding studiesconfirmed that each of these IgG format r84 antibodies also binds toboth murine VEGF and human VEGF (FIG. 19). These results show anotheradvantage of the selected fully human r84 antibody over the 2C3antibody, as 2C3 does not exhibit meaningful binding to murine VEGF.

The absence of meaningful binding of 2C3 to murine VEGF has beendemonstrated in an indirect ELISA assay. In this assay, the interactionof 2C3 with human and mouse VEGF as well as other VEGF family memberswas assessed.

The indirect ELISA assays were performed essentially as described inBrekken et al., Cancer Research 1998 and 2000. Briefly, the variousgrowth factors, i.e. human VEGF-A (VEGF), mouse VEGF, PIGF, VEGF-B,VEGF-C and VEGF-D, were purchased from R&D Systems and coated onto thewells of an ELISA plate (50 μl/well at 0.5 g/ml in sensitizing buffer,overnight at 4° C.). The wells were blocked in 5% CAH (casein acidhydrolysate, Sigma, made up in PBS) for 1 hr at 37° C. and incubated intriplicate with the 2C3 anti-VEGF antibody at 1.0 μg/ml for 2 hours atroom temperature. Binding was detected with peroxidase-conjugatedsecondary antibody (either anti-human or anti-mouse IgG, diluted1:5000). The wells were developed with TMB (a colorimetric substrate forHRP) and absorbance read at 450 nM. The mean absorbance values are asfollows: human VEGF-A (3.07), mouse VEGF (0.09) which was the same asthe background signal, PIGF (0.1), VEGF-B (0.09), VEGF-C (0.09) andVEGF-D (0.12).

The results demonstrate that 2C3 binds to human VEGF-A but does notreact with mouse VEGF-A, PIGF, VEGF-B, VEGF-C, or VEGF-D. This assay hasbeen replicated several times with similar results.

Further evidence that the IgG form of the r84/PGN311 antibody binds tomouse VEGF, whereas 2C3 and Avastin do not bind to mouse VEGF has beenobtained in experiments in which mouse VEGF levels in serum have beenevaluated in animals treated with r84, 2C3 and Avastin.

Sera from tumor-bearing animals treated with a control IgG (Synagis),avastin, 2C3 or r84 was collected and assayed by ELISA for the level ofmouse VEGF using a kit from R&D systems. In addition, some samples ofsera from r84 treated mice were immunodepleted of all antibodies byincubation with protein G beads.

The results are shown in FIG. 24. The serum level of mouse VEGF is verysimilar between control, avastin and 2C3 treated animals. However, theserum level of mouse VEGF is dramatically higher in animals treated withr84. The difference in level of mouse VEGF between that observed withthe control, avastin and 2C3 treated animals and that observed with ther84 treated animals is evidence that the control, avastin and 2C3antibodies do not bind to mouse VEGF, whereas the r84 IgG antibody doesbind to mouse VEGF.

The “r84” column in FIG. 24 shows the total amount of VEGF in the sera(i.e. free (biologically active) VEGF and VEGF complexed with r84). Itis believed that the amount of free (biologically active) VEGF is animportant parameter to be measured when anti-VEGF antibodies are usedtherapeutically, in particular to assess the effectiveness of theantibody at binding to VEGF (Loupakis et al., 2007). The “r84 supe”column shows the amount of free VEGF in the sera after the r84immunoglobulin and r84 bound to murine VEGF were removed by incubationwith protein G. FIG. 24 thus shows that free mouse VEGF levels in theserum of r84 treated animals are at baseline levels. Thus, the resultsin FIG. 24 not only demonstrate that r84 binds well to mouse VEGF butalso demonstrate that r84 is very effective at depleting levels of free(biologically active) VEGF in serum, which is an important property foruse in therapy.

The results discussed in Example 11E below using a syngeneic mousemammary tumor model and showing that mouse chimeric r84 significantlyimproved the survival of tumor bearing mice is further validation thatr84 binds and blocks mouse VEGF activity in vivo.

The results described above, show that the fully human r84/PGN311antibody binds to both murine and human VEGF, whereas the 2C3 andAvastin antibodies do not bind to murine VEGF. This is an importantadvantage in terms of being able to use r84 to assess for example antitumor activity both in mouse syngeneic models, i.e. where mouse tumorcells are administered to mice and in xenograft models, i.e. where humantumor cells are administered to mice.

In addition, the ability to bind both mouse and human VEGF as shown byr84/PGN311 but not by antibodies such as 2C3 and Avastin, means that theresults shown by r84 in xenograft mouse models are more likely to berepresentative of the activity of r84 in human subjects, i.e. theresults with r84 in pre-clinical mouse models are likely to be a goodmodel for what will be seen when the antibody is put into patients. Thereason for this is that antibodies which can only bind to human VEGF(e.g. Avastin and 2C3) will bind to VEGF produced by the human tumorcells but will not be able to bind to endogenous murine VEGF. This is ofcourse unlike the situation in a human patient, in which VEGF producedby the tumor and endogenous VEGF would be present.

The potential disadvantage with such a situation is that an antibodywhich binds to human VEGF but not mouse VEGF might perform well in amouse xenograft model but this would not be reflected by a similarperformance in a human system where much more VEGF was present. In otherwords, the anti-tumor effect seen in a mouse xenograft system with anantibody which can only bind to human VEGF might look better than theclinical reality. In contrast, if you are working with an antibody thatcan bind to both human and mouse VEGF then this will bind to all formsof VEGF present in the mouse model system and is likely to be much morerepresentative of the situation when the antibody is put into humans.This is an important advantage and one which is displayed by theantibodies of the invention.

Example 5 Binding Affinity of EJ173/112-C11 (r84/PGN311)

Biacore was used to assess the binding affinity of various antibodies.Different scFv antibodies at a concentration of 1 μM (micromolar) wereflown over a CM5 chip with immobilized VEGF (amine coupling). Thebinding curves are shown in FIG. 5, where it can be seen that the scFvform of r84/PGN311 has a noticeably higher binding affinity than thesingle chain form of the mother clone (m).

In addition, initial studies were carried out to calculate the bindingaffinity of r84 IgG for VEGF, in which various concentrations of r84 IgGwere flown over immobilized VEGF-A. In this regard, the bindingaffinity, expressed as the K_(d), was calculated using the 1:1 bindingmodel in the Biacore 3000 Evaluation software. The K_(d) value obtainedfor r84 IgG in this initial study was calculated as 6.7×10⁻⁹ M.

Subsequent affinity studies using BiaCore have yielded the affinity datashown in Table 2. For these experiments, the affinity of the IgG formatsof EJ173/112-C11 (r84/PGN311) and 2C3 were determined by immobilizing100 RU of human VEGF-A on a CM5 chip (Biacore). A dilution series (100nM and 2-fold dilutions) of each IgG was flown over the VEGF-coated flowcell at a flow rate of 50 μl/min. The background signal from a flow cellcoated with BSA was subtracted from the binding curves. The bindingaffinity expressed as the K_(D) was calculated by the 1:1 Fitting modelusing software belonging to the Biacore T100 machine.

TABLE 2 Coated VEGF Coated VEGF KD (M) (100 RU), 25° C. (100 RU), 37° C.2C3 1.24 × 10⁻⁸ 3.13 × 10⁻⁷ r84 3.21 × 10⁻⁹ 5.22 × 10⁻⁹

The data shows that r84 binds to VEGF with superior affinity than 2C3 atboth 25° C. and 37° C. It is reasonable to expect that this differencewill lead to superior characteristics of r84 when compared to 2C3 inmany clinical settings related to the treatment of angiogenic diseases,including cancer. The results at 37° C. are particularly interesting asthis is the temperature the antibodies will be subjected to when used invivo. It should also be noted, that in this experiment, Avastin showedan about 10 fold loss in affinity when comparing binding at 25° C. and37° C., while r84 is less sensitive to temperature (reduction of KD noteven 2-fold).

Example 6 Conversion of the scFv Form of r84/PGN311 to IgG Forms

A fully human IgG form of r84 was first constructed, as follows. The VHand VL chains of the scFv form of the r84/PGN311 antibody sequence asshown in FIG. 1 were taken and inserted into Lonza pCon IgG1a and kappaexpression vectors and then combined to create one vector containing theentire r84 antibody gene. To make the full length IgG antibody, thevector was then transfected into CHO K1SV cells.

Once the growth conditions were optimized, the production rate of thecell line was approximately 5 milligrams of antibody per liter of cellculture. Although this expression method was effective, not all of thepurified antibody was stable. After buffer optimization, the aggregationof the antibody was reduced, achieving 89.5% monomer and 10.5%aggregate.

The optimized growth conditions used are Invitrogen CD-CHO with 40 μMMSX, pH 6.8-7.0, 5% CO₂, 37° C. The optimized buffer used is 10 mMsodium phosphate, 25 mM sodium acetate, 50 mM Glycine at pH 5.5.

In order to further increase the stability of r84/PGN311, the amino acidsequence was analyzed. Comparing the r84 sequence to typical humanantibody sequences indicated that the last amino acid of the VL chain ofr84 was missing from the construct being used (i.e., the last Lysineresidue, K, was missing). This residue was reintroduced. The DNAsequence was also changed (without changing the translated amino acidsequence) to be more “compatible” with CHO cells. The result was a newDNA sequence, and an amino acid sequence in which the VL chain endedwith Lysine. The nucleotide and amino acid sequences of the new completeheavy and light chains of the IgG antibody are shown below:

r84/PGN311 IgG Heavy chain (nucleic acid sequence) (SEQ ID NO: 22)CAGGTACAGCTTGTGCAGTCCGGAGCCGAGGTGAAGAAACCCGGAGCATCAGTGAAGGTTAGCTGCAAGGCATCTGGTGGGACATTTTCCTCCTATGCCATCTCCTGGGTTCGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGGGGGTTCGATCCCGAAGACGGAGAGACCATTTACGCACAGAAGTTTCAGGGTCGCGTGACCATGACCGAGGATACTTCTACCGACACAGCATATATGGAGCTCAGTAGCTTGCGCTCCGAGGACACGGCTGTATATTACTGTGCCACTGGACGGAGCATGGTGCGCGGGGTAATCATCCCTTTCAACGGGATGGATGTATGGGGCCAAGGGACCACCGTGACAGTCAGCTCTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAGr84/PGN311 IgG Heavy chain (amino acid sequence) (SEQ ID NO: 24)QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGRSMVRGVIIPFNGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSTCVVVDVSHEDPEVLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKr84/PGN311 IgG Light chain (nucleic acid sequence) (SEQ ID NO: 23)GACATTCGGATGACTCAGTCTCCCTCCTCTTTGAGCGCTTCTGTGGGCGATAGGGTTACTATCACTTGTCGAGCCTCTCAATCCATCAGCTCCTACTTGAACTGGTACCAGCAGAAACCCGGGAAAGCACCCAAGCTGCTTATTTACGCCGCCTCCTCCCTGCAATCCGGAGTGCCCTCCCGTTCAGCGGCTCCGGCTCTGGAACAGACTTTACCCTGACCATTTCTTCTTTGCAGCCTGAGGATTTTGCTACTTACTACTGTCAGCAGAGTTACTCCACCCCTTTGACATTCGGTGGTGGAACGAAAGTAGAAATTAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGr84/PGN311 IgG Light chain (amino acid sequence) (SEQ ID NO: 25)DIRMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDPATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNPYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The new r84/PGN311 DNA sequences shown above were inserted into LonzapCon IgG1a and kappa expression vectors and then combined to create onevector containing the entire r84/PGN311 antibody sequence. The vectorwas then transfected into CHO K₁SV cells. Antibody production wasincreased to over 350 milligrams per liter with little cell cultureoptimization. Even more importantly, the antibody was much more stable,with monomer over 99%.

The nucleic acid sequences of the variable heavy and variable lightchains of the IgG form of r84/PGN311 are also shown below:

r84/PGN311 IgG VH chain (nucleic acid sequence) (SEQ ID NO: 26)CAGGTACAGCTTGTGCAGTCCGGAGCCGAGGTGAAGAAACCCGAGCATCAGTGAAGGTTAGCTGCAAGGCATCTGGTGGGACATTTTCCTCCTATGCCATCTCCTGGGTTCGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGGGGGTTCGATCCCGAAGACGGAGAGACCATTTACGCACAGAAGTTTCAGGGTCGCGTGACCATGACCGAGGATACTTCTACCGACACAGCATATATGGAGCTCAGTAGCTTGCGCTCCGAGGACACGGCTGTATATTACTGTGCCACTGGACGGAGCATGGTGCGCGGGGTAATCATCCCTTTCAACGGGATGGATGTATGGGGCCAAGGGACCACCGTGACAGTCAGCTCTr84/PGN311 IgG VL chain (nucleic acid sequence) (SEQ ID NO: 27)GACATTCGGATGACTCAGTCTCCCTCCTCTTTGAGCGCTTCTGTGGGCGATAGGGTTACTATCACTTGTCGAGCCTCTCAATCCATCAGCTCCTACTTGAACTGGTACCAGCAGAAACCCGGAAAGCACCCAAGCTGCTTATTTACGCCGCCTCCTCCCTGCAATCCGGAGTGCCCTCCCGGTTCAGCGGCTCCGGCTCTGGAACAGACTTTACCCTGACCATTTCTTCTTTGCAGCCTGAGGATTTTGCTACTTACTACTGTCAGCAGAGTTACTCCACCCCTTTGACATTCGGTGGTGGAACGAAAGTAGAAATTAAG

A murine chimeric version of the r84 antibody was also constructed. Thiswas performed to attach the fully human variable region to a mouseconstant region, providing a mouse antibody for use in certainpreclinical studies in mice. The generation of the murine chimericantibody was performed as described above for the fully human IgG, butattaching a nucleic acid sequence encoding the fully human variableregion to a nucleic acid sequence encoding a mouse constant region.

The nucleotide and amino acid sequences of the new complete heavy andlight chains of the murine chimeric IgG antibody are shown below:

R84/PGN 311 Chimeric Heavy Chain (nucleic acid sequence) (SEQ ID NO: 31)CAGGTGCAGCTGGTGCAATCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGTTTTGATCCTGAAGATGGTGAAACAATCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCGAGGACACATCTACAGACACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCAACAGGACGTTCTATGGTTCGGGGAGTCATTATACCTTTTAACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCACGCGCCGATGCTGCACCGACTGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGAR84/PGN 311 Chimeric Heavy Chain (amino acid sequence) (SEQ ID NO: 32)QVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATGRSMVRGVIIPFNGMDVWGQGTTVTVSSRADAAPTVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKR84/PGN 311 Chimeric Light Chain (nucleic acid sequence) (SEQ ID NO: 33)GATATCAGGATGACGCAGAGTCCAAGCTCTCTGTCTGCCTCTGTGGGGGACAGGGTGACTATTACTTGTCGGGCATCACAGAGTATCTCCAGCTACCTTAATTGGTACCAGCAAAAGCCCGGCAAAGCCCCCAAATTGCTGATTTACGCAGCCAGCTCCCTTCAGTCTGGCGTCCCTAGCCGCTTCTCCGGGAGCGGATCAGGCACAGACTTTACGTTGACAATCAGTTCTCTGCAGCCGGAGGATTTTGCCACTTACTACTGTCAACAGAGCTACAGTACGCCTCTCACGTTTGGCGGTGGGACAAAGGTGGAAATCAAACGGGCTGATGCTGCACCGACTGTGTCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTR84/PGN 311 Chimeric Light Chain (amino acid sequence) (SEQ ID NO: 34)DIRMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

Example 7 EJ173/112-C11 (r84/PGN311) Inhibits VEGFR2-Mediated Events

A. r84/PGN311 Inhibits Cell Signaling via VEGFR2

Cell assays were used to confirm the cellular performance of theselected antibody. VEGF stimulates intracellular signaling through theMAPK kinase pathway, which also involves the activation (throughphosphorylation) of two proteins called MAPK1 and MAPK2, at 44 and 42kilo Daltons, respectively. Another name for these proteins is Erk1 andErk2. Antibodies that inhibit the interaction of VEGF with VEGFR2 caninhibit Erk1/2 activation as well.

bEnd.3 or PAE/KDR cell lines (obtained from Dr. Philip Thorpe atUT-Southwestern Medical Center, Dallas, Tex. and Dr. JohannesWaltenberger, Ulm University Medical Center, Ulm, Germany, respectively)express VEGFR2 on their surface, allowing them to be stimulated withVEGF. The cell lines were starved 48 hours for serum and growth factors,and next stimulated through the addition of 10 (V10) and 50 ng/mlVEGF165 (V50) in the presence or absence of the candidate in IgG formatat 4 μg/ml. Non stimulated cells were the negative control (NS). Afterthis stimulation, the cells were washed with ice cold PBS containingdifferent phosphatase inhibitors before they were lysed. The centrifugedcell extract was run on a polyacrylamide gel, and the separated proteinswere subsequently blotted to a nitrocellulose membrane. The blot wasprobed with an anti-phospho Erk1/2 antibody to monitor the inhibition ofErk1/2 phosphorylation (FIG. 6). Total Erk1/2 was also detected as aninternal control of cell levels. The r84/PGN311 IgG clone inhibitedphosphorylation of Erk1/2.

VEGF also stimulates intracellular signaling through the phospholipaseCγ (PLC-γ) pathway, which involves the activation (throughphosphorylation) of protein at 155 kilo Daltons. Antibodies that inhibitthe interaction of VEGF with VEGFR2 can inhibit PLC-γ activation aswell.

Human dermal microvascular endothelial cell lines (HDMEC, Lonza, catalog#CC2810) express VEGFR2 on their surface allowing them to be stimulatedwith VEGF. HDMECs were plated at 250,000 cells per well using 6-wellplates. Cells were allowed to adhere overnight in 5% FBS ECM(endothelial cell media). The cells were then serum starved for 24 hoursand stimulated through the addition of 50 ng/ml VEGF165 (+VEGF) in thepresence of the antibodies, Avastin (bevacizumab, Presta et al., 1997),r84/PGN311, 2C3 (all in IgG format) or a control IgG antibody (Synagis(palivizumab), human anti-RSV, MedImmune). The IgGs were used at 90μg/ml. Non stimulated cells were the negative control (NT). Avastin andr84/PGN311 were also added to cells in the absence of VEGF.

After this stimulation, the cells were washed with ice cold PBScontaining different phosphatase inhibitors before they were lysed. Thecentrifuged cell extract was run on a polyacrylamide gel, and theseparated proteins were subsequently transferred to a nitrocellulosemembrane. The blots were probed with an anti-phospho Erk1/2 antibody(FIG. 16A, pERK1/2) and anti-phospho PLC-γ antibody (FIG. 16A, pPLC-γ)to monitor the inhibition of phosphorylation. Total Erk1/2 (FIG. 16A,ERK1/2) and PLC-γ (FIG. 16A, PLC-γ) were also detected as an internalcontrol of cell levels. The expression of VEGFR2 on the HDMECs wasdetected with an antibody to VEGFR2 (FIG. 16A). FIG. 16A shows that ther84/PGN311 IgG antibody inhibited phosphorylation of both Erk1/2 andPLC-γ.

Using the same methodology as described above, PAE Flt1 cells expressingVEGFR1 were treated and starved in the same way before being leftuntreated or being stimulated through the addition of 50 ng/ml VEGF165in the presence or absence of the antibodies, Avastin or r84/PGN311 (inIgG format). Blots were prepared and probed with an anti-phospho VEGFR1antibody (FIG. 16B, phospho VEGFR1). Total VEGFR1 (FIG. 16B, totalVEGFR1) was also detected as an internal control of cell levels. Thedata in FIG. 16B show that r84/PGN311 did not inhibit phosphorylation ofVEGFR1, whereas the positive control, Avastin, did inhibitphosphorylation of VEGFR1. Together, FIG. 16A and FIG. 16B confirm thatr84/PGN311 selectively blocks the VEGFR2 pathway.

B. r84/PGN311 Blocks VEGF-Induced Migration of VEGFR2-Expressing Cells

As expected, it was also confirmed that r84/PGN311 is able to potentlyinhibit VEGF-induced migration of VEGFR2-expressing endothelial cells.Examples of this activity are shown in FIG. 20A for HDMEC, and in FIG.20B for PAE KDR-expressing cells. In each of these assays, it will benoted that r84 significantly inhibits VEGF-induced migration ofVEGFR2-expressing cells and performs at least as well as Avastin.Comparative studies using VEGFR1-expressing PAE Flt1 cells showed thatr84 does not inhibit VEGF-induced migration of VEGFR1-expressing cells(FIG. 21). In contrast, Avastin significantly inhibits VEGF-inducedmigration of VEGFR1-expressing cells (FIG. 21).

Example 8 EJ173/112-C11 (r84/PGN311) Binds to VEGF121

The binding of EJ173/112-C11 (r84/PGN311) scFv to a biologically activeisoform of VEGF-A, VEGF121, was determined (R&D Systems HuVEGF121298-VS-005/CF). 2 μg/ml of carrier free VEGF121 was plated onpolystyrene immunoplates. 10 μg/ml of purified scFv was added anddetected with an anti-c-myc tag mouse monoclonal antibody (Invitrogen)and HRP-conjugated secondary rabbit anti-mouse antibody.

The results showed that EJ173/112-C11 (r84/PGN311) (FIG. 7) was positivefor VEGF121. The B9 murine scFv control recognized VEGF121, but at alower level than the human variants.

Example 9 EJ173/112-C11 (r84/PGN311) Blocks VEGF Binding to VEGFR2 butnot VEGFR1

96-well ELISA plates (BD Falcon, cat #353279) were coated overnight at4° C. with soluble HuVEGFR1/Fc (R&D Systems, cat #321-FL-050, CF) orHuVEGFR2 (R&D Systems 357-KD-050/CF) at a concentration of 1.0 μg/ml in50 μl of sensitizing buffer/well. The wells were washed in wash buffer(WB) (TBSt (Tris buffer Saline, 0.1% Tween 20)) and blocked for 1 hr at37° C. in 20% Aquablock (East Coast Biologics, Inc.) in WB. 50 μl of IgGat the appropriate concentration or WB was added to the wells followedimmediately by VEGF-biotin at a final concentration of 100 ng/ml. Theplate was incubated for 2 hr at RT, washed 3× and incubated for 1 hr atRT with 100 μl/well of peroxidase-conjugated avidin (JacksonImmunoResearch) at a 1:7500 dilution in WB. After washing the plates 4×,signal was developed with TMB, stopped with acid and read at 450 nM.

The results of these assays are shown in FIG. 8A and FIG. 8B. The signalof VEGF alone (VEGF) or VEGF in the presence of the indicated antibodywas normalized to VEGF alone (100%). The mean+/−SEM is shown. N=12 (4identical plates with each treatment performed in triplicate). A signalof less than 50% is considered significant inhibition of binding.

As shown in FIG. 8A and FIG. 8B, the results from this controlled studyshow that r84/PGN311 substantially blocks the interaction of VEGF withVEGFR2, but does not substantially block the interaction of VEGF withVEGFR1. Parallel studies using r84/PGN311 and Avastin (bevacizumab)(Presta et al., 1997) confirmed the known properties of Avastin assubstantially blocking the interaction of VEGF with both VEGFR2 andVEGFR1. In addition, parallel studies using r84/PGN311 and the originalmurine 2C3 antibody showed that the differential in substantiallyblocking VEGF binding to VEGFR2, but not substantially blocking VEGFbinding to VEGFR1, was even greater for r84 than for 2C3. This showsanother surprising advantage of r84 over the 2C3 antibody.

Example 10 Tumor Associated Macrophages Express VEGFR2

A. Model

Orthotopic tumors were established in athymic nude mice (7-9 weeks) byinjecting 1×10⁶ MiaPaCa-2 cells into the tail of the pancreas. Tumorswere allowed to develop for one week prior to initiating therapy.

B. Treatment

Animals were treated with control antibody (C44), or the murine 2C3antibody via i.p. injection twice a week for three weeks. At sacrifice,tumors were excised and with the residual pancreas, weighed and weresnap frozen or fixed in methyl carnoys for histochemical andimmunohistochemical analysis.

C. IHC

Antibodies used for macrophage markers include CD86, CD14 and F4/80.VEGFR2 antibodies T014 and RAFL-2 were used.

D. Peritoneal Macrophage Isolation

Macrophages from tumor-bearing (TB) or non-tumor bearing (NTB) animalswere isolated by sterile peritoneal lavage.

E. Results

Surprisingly, it was found that tumor associated macrophages (TAM)expressed VEGFR2. This explains the observation that 2C3 reducedinfiltration of VEGFR2-positive TAM in vivo. The results are shown inFIGS. 10A, B and C.

In this regard, FIG. 10A depicts co-localization of T014 (VEGFR2antibody) and F4/80 (macrophage marker) staining on tumor sections fromcontrol treated or 2C3 treated animals. 2C3 decreases macrophageinfiltration. However, both groups demonstrate co-localization of VEGFR2and macrophage markers. The number of cells double positive for one ofthree different macrophage markers and VEGFR2 is depicted in FIG. 10B.In FIG. 10C, peritoneal macrophages from tumor bearing animalsdemonstrate VEGFR2 using two different antibodies.

Example 11 r84/PGN311 Decreases Tumor Volume in Animals

Animal models were used to show that administration of the r84/PGN311antibody leads to a significant reduction in tumor volumes.

A. MDA-MB-231 Breast Cancer Cell Tumor Model

5 million MDA-MB-231 cells were injected into the mammary fat pad ofSCID mice. Tumors developed for 26 days prior to the start of therapy.At this time, animals were randomly assigned to treatment groups. 100 μlof saline (control, n=5) or 250 μg in 100 μl of buffer of Avastin IgG(n=8) or r84/PGN311 IgG (n=9) was given by subcutaneous injection 2× perweek. The results are shown in FIG. 11, which displays mean tumorvolume+/−SEM. Avastin and r84 treated mice have tumor volumes that aresignificantly smaller than control treated animals. FIG. 12 displaystumor weight/body weight for individual animals in each group. Avastinand r84 treated mice have tumor/body ratios that are significantlysmaller than control treated animals. The results in FIG. 11 and FIG. 12show that r84 performs essentially as effectively as Avastin.

B. A673 Rhabdomyosarcoma Tumor Model

x10⁶ A-673 cells (a human rhabdomyosarcoma cell line—ATCC CRL-1598) wereinjected subcutaneously into 22 nu/nu (NCI) mice. The mice were dividedinto 3 groups (n=8/grp for 2C3 and r84/PGN311, n=6/control grp) andtherapy was initiated 5 days post tumor cell injection (TCI). Therapyconsisted of 50 μg of the indicated IgG injected ip 2×/week. Animalweight and tumor volume was monitored. The control antibody was Synagis(human anti-RSV).

The mice received 8 injections of therapy between day 5 and day 29 postTCI. FIG. 13 shows the tumor volume of each group on day 30 post TCI.One-way ANOVA indicates that the groups are statistically different;furthermore 2C3 and r84/PGN311 are different from control by “Dunnett'sMultiple Comparison Test” (p<0.05). It is clear from these results that2C3 and r84 reduced tumor growth at the modest dose of 50 μg/injection2×/week. r84 displayed activity that was essentially the same as 2C3.The overall conclusion from this animal study is that 2C3 and r84 areeffective at controlling the growth of A673 tumors.

C. Human Non-Small Cell Lung Cancer (NSCLC) Models

Further in vivo animal models were used to show that administration ofthe r84/PGN311 antibody leads to significant reductions in tumor growth.

The ability of r84/PGN311 to inhibit tumor growth in vivo was tested infour different human non-small cell lung cancers (NSCLC), H1299 (ATCCCRL-5803), H460 (ATCC HTB-177), H358 (ATCC CRL-5807) and A549 (ATCCCCL-185). SCID mice (n=25 per cell line) were injected subcutaneouslywith 2.5×10⁶ cells and therapy was initiated 1 day post TCI. Therapyconsisted of 250 μg of Synagis or XTL (negative control), Avastin IgG(positive control) or 500 μg of r84/PGN311 IgG delivered intraperitoneally (i.p.) 2 times each week. Therapy was given until the micewere sacrificed. The H460 mice were sacrificed after 40 days, the H1299mice were sacrificed after 48 days, the A549 mice were sacrificed after55 days, and the H358 mice were sacrificed after 83 days. The tumorweights were measured, and the results are shown in FIG. 17A (for H460),FIG. 17B (for H1299), FIG. 17C (for H358) and FIG. 17D (for A549) asmean tumor weight+/−SEM. The ratios of the treated tumor weights overthe control tumor weights are also shown in FIG. 17A, FIG. 17B, FIG. 17Cand FIG. 17D (“T/C”).

FIG. 17A, FIG. 17B, FIG. 17C and FIG. 17D show that the Avastin andr84/PGN311 treated animals have tumor weights that are significantlysmaller than control treated animals. r84/PGN311 performs better thanAvastin in the H460 (FIG. 17A), H1299 (FIG. 17B) and the A549 (FIG. 17D)models. r84/PGN311 performs at least as effectively as Avastin in theH358 assay (FIG. 17C).

D. Panel Pancreatic Cancer Cell Tumor Model

Mice bearing pancreatic adenocarcinoma cells, Panel, were given eitherr84/PGN311 IgG or Synagis as a negative control. Therapy was given untilthe mice were sacrificed. The tumor volumes were measured and theresults are shown in FIG. 22. It can be seen that r84/PGN311 markedlyreduces Panel tumor growth in comparison to control (FIG. 22).

E. 4T1 Mammary Tumor Model

Controlled studies using the mouse chimeric version of r84/PGN311 showedthat the r84 antibody is able to prolong the survival of mice bearingsyngeneic 4T1 mammary tumors. As shown in FIG. 23, treatment with mousechimeric r84/PGN311 resulted in the mice surviving longer than theanimals in the control treated group.

Example 12 r84/PGN311 Reduces Microvessel Density and Infiltration ofTumor Associated Macrophages

Tumors were taken from the mice used in the MDA-MB-231 animal modelstudy described in Example 11 and also from mice which had been treatedin parallel in accordance with the same regimen with 250 μg in 100 μl ofbuffer of 2C3 IgG. These tumors were sectioned and stained withantibodies to mouse endothelial cells (MECA-32) and to a macrophagemarker (Mac-3). Three tumors from control animals and three tumors eachfrom r84/PGN311 and 2C3 treated animals were analyzed and 5 images fromeach tumor were studied. The results are shown in FIG. 14 and FIG. 15,which show that tumors from r84 and 2C3 treated animals showedsignificantly reduced number of blood vessels/high power field (MECA-32,p<0.0001, FIG. 15) and significantly reduced expression of themacrophage marker (Mac-3, p<0.01 for r84, FIG. 14). This is evidencethat r84 significantly reduces microvessel density and infiltration oftumor associated macrophages and that r84 has a more pronounced effectthan 2C3 on reducing the infiltration of tumor associated macrophages(FIG. 14).

Example 13 Effects of r84/PGN311 on Cells Infiltrating Tumors

A. Polymorphonuclear Leukocytes

Tumors were taken from the mice used in the MDA-MB-231 animal modelstudy described in Example 11 and also from mice which had been treatedin parallel in accordance with the same regimen with 250 μg in 100 μl ofbuffer of 2C3 IgG. The study has shown that tumors from r84 and 2C3treated animals have significantly increased polymorphonuclear leukocyte(PMN) infiltration in comparison to control. This effect wasstatistically significant for the r84 and 2C3 antibodies. AlthoughAvastin (bevacizumab) also increased PMN infiltration into MDA-MB-231tumors in comparison to control, in contrast to r84 and 2C3, thisincrease was not statistically significant for Avastin.

B. CD11b+/Gr1+ Cells

Further studies in MDA-MB-231 tumor-bearing mice have shown thatsignificantly less CD11b/Gr1 double positive cells infiltrate the tumorsin r84-treated animals as opposed to control. In comparative studies,neither the 2C3 antibody nor Avastin showed a statistically significantdecrease in CD11b+/Gr1+ infiltration, although some reduction wasmeasurable in Avastin-treated animals.

Tumors were taken from the mice used in the MDA-MB-231 animal modelstudy described in Example 11 and also from mice which had been treatedin parallel in accordance with the same regimen with 250 μg in 100 μl ofbuffer of 2C3 IgG. The study has shown that significantly lessCD11b+/Gr1+ double positive cells infiltrate the tumors in r84-treatedanimals as opposed to control (as assessed by ANOVA, p<0.01, shown by **in FIG. 25). The decrease in the number of double-positive cells was39%. In comparative studies, 2C3 did not show a statisticallysignificant decrease in CD11b+/Gr1+ infiltration (FIG. 25).

The reduced infiltration of myeloid derived suppressor cells CD11b+/Gr1+is of special interest, as cells expressing both markers have recentlybeen associated with mediation of tumor refractoriness to anti-VEGFtherapy (Shojaei et al., 2007). Myeloid-derived suppressor cells(CD11b+Gr1+) are also an important contributor to tumor progression. Inthe tumor microenvironment these cells secrete immunosuppressivemediators and induce T-lymphocyte dysfunction (Gabrilovich et al., 2001;Serafini et al., 2004).

As the tumor infiltration of CD11b+/Gr1+ cells is leastpronounced/significantly lower in the r84-treated animals, it suggeststhat treatment with r84 is less prone to the development of drugresistance or refractoriness to anti-VEGF therapy than treatment withother drugs targeting VEGF.

This ability to reduce infiltration of CD11b+/Gr1+ cells into tumors isthus a further advantageous property shown by the r84/PGN311 antibodyand is a property which has a potential importance for therapeuticapplications of r84/PGN311. Furthermore, this property is not shown bythe 2C3 antibody and only at a more reduced level by Avastin.

Example 14 r84/PGN311 Reduces Lymphatic Density in Tumors

Mice with MDA-MB-231 tumors were treated with r84/PGN311 or controlantibody and tumor sections analyzed to show the lymphatic vesselswithin the tumors. The results are presented in FIG. 18A, FIG. 18B andFIG. 18C, which show that the lymphatic vessel density in r84-treatedtumors is significantly lower than in control tumors.

In particular, immunofluorescence staining of frozen MDA-MB-231 tumorsections was first performed to identify lymphatic vessels via thelymphatic markers, podoplanin and Prox1. These results are set forth inFIG. 18A, which shows podoplanin (green), Prox1 (red) and the mergedimages, thus identifying lymphatic vessels in control (top panels) andr84-treated tumors (bottom panels). LYVE-1 staining was also performedin consecutive MDA-MB-231 tumor sections. As shown in FIG. 18B, theseresults indicate that the pattern of lymphatic vessels in MDA-MB-231tumor sections stained for LYVE-1 is similar to that observed forpodoplanin and Prox1 (FIG. 18A).

To determine whether the density of lymphatic vessels in control andr84-treated tumors was different, the entire area of each LYVE-1 stainedtumor section was examined at low magnification and the percent ofLYVE-1 positive area was determined for each field using NIS-Elementsimaging software. The ten fields with the highest LYVE-1 positivepercent area were averaged together to yield a final score for eachtumor and group means were tested for significance by an unpairedstudent's t-test. As depicted in FIG. 18C, the percent of LYVE-1positive area of control tumors (7.03±1.013; n=6) was significantlygreater than r84 treated tumors (2.23±0.986; n=5), with P=0.0042. Theseresults, showing that treatment with r84/PGN311 significantly lowerstumor lymphatic vessel density, thus support the use of the humanantibodies of the invention to inhibit lymphangiogenesis.

Example 15 Chronic Administration of r84/PGN311 does not Induce Toxicityin Mice

Non-tumor bearing and tumor-bearing mice were used in these studies.

5×10⁶ Panc-1 cells (human pancreatic cancer cell line, ATCC CRL-1469),were injected into 10 SCID mice. On Day 1 post TCI, 5 tumor-bearing and5 non-tumor bearing mice were injected i.p. with 500 μg of Synagis orr84/PGN311 IgG. Therapy was given by injection 2 times per week and wascontinued for 12 weeks, after which the mice were sacrificed. At thetime of sacrifice blood was taken and analyzed for standard bloodchemistry. The liver, kidney, and thyroid were also harvested forhistological evaluation.

These analyses showed that there were no overt changes in histology ofany tissue examined by H&E staining (this examination was carried out ina blinded fashion by a pathologist who specializes in mouse tissuehistology). Furthermore, blood chemistry was analyzed for 22 differentanalytes and no significant changes between control, naïve mice, or r84treated animals were found.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the composition, methodsand in the steps or in the sequence of steps of the methods describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentsthat are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Abrams and Oldham, “In: Monoclonal Antibody Therapy of Human    Cancer”, Foon and Morgan (Eds.), Martinus Nijhoff Publishing,    Boston, 103-120, 1985.-   Aiello, Pierce, Foley, Takagi, Chen, Riddle, Ferrara, King, Smith,    “Suppression of retinal neovascularization in vivo by inhibition of    vascular endothelial growth factor (VEGF) using soluble    VEGF-receptor chimeric proteins,” Proc. Natl. Acad. Sci., USA,    92:10457-10461, 1995.-   Alderson, McGowan, Baldridge, Probst, “TLR4Agonists as    Immunomodulatory Agents”, J. Endoxin Res., 12(5):313-319, 2006.    Alon, Hemo, Inn, Pe'er, Stone, Keshet, Vascular endothelial growth    factor acts as a survival factor for newly formed retinal vessels    and has implications for retinopathy of prematurity,” Nature Med.,    1:1024-1028, 1995.-   Altschul, Madden, Schaffer, Zhang, Zhang, Miller, Lipman, “Gapped    BLAST and PSI-BLAST: a new generation of protein database search    programs”, Nucleic Acids Res., 25:3389-3402, 1997.-   Anthony, Wheeler, Elcock, Pickett, Thomas, “Short report:    identification of a specific pattern of vascular endothelial growth    factor mRNA expression in human placenta and cultured placental    fibroblasts”, Placenta, 15:557-61, 1994.-   Apetoh, Ghiringhelli, Tesniere, Obeid, Ortiz, Criollo, Mignot,    Maiuri, Ullrich, Saulnier, Yang, Amigorena, Ryffel, Barrat, Saftig,    Levi, Lidereau, Nogues, Mira, Chompret, Joulin, Clavel-Chapelon,    Bourhis, Andre, Delaloge, Tursz, Kroemer, Zitvogel, “Toll-Like    Receptor 4-Dependent Contribution of the Immune System to Anticancer    Chemotherapy and Radiotherapy”, Nat. Med., 13:1050-1059, 2007.-   Arbabi-Ghahroudi, Desmyter, Wyns, Hamers, Muyldermans, “Selection    and identification of single domain antibody fragments from camel    heavy-chain antibodies”, FEBS Lett., 414:521-526, 1997.-   Asahara, Murohara, Sullivan, Silver, van der Zee, Li, Witzenbichler,    Schatteman, Isner, “Isolation of putative progenitor endothelial    cells for angiogenesis,” Science, 275(5302):964-967, 1997.-   Asano, Yukita, Matsumoto, Kondo, Suzuki, “Inhibition of tumor growth    and metastasis by an immunoneutralizing monoclonal antibody to human    vascular endothelial growth factor/vascular permeability factor,”    Cancer Res., 55:5296-5301, 1995.-   Asano, Yukita, Matsumoto, Hanatani, Suzuki, “An anti-human VEGF    monoclonal antibody, MV833, that exhibits potent anti-tumor activity    in vivo,” Hybridoma, 17:185-90, 1998.-   Baca, Presta, O'Connor, Wells, “Antibody humanization using    monovalent phage display,” J. Biol. Chem., 272(16):10678-84, 1997.-   Baldari, Murray, Ghiara, Cesareni, Galeotti, “A Novel Leader Peptide    Which Allows Efficient Secretion of a Fragment of Human Interleukin    1 Beta in Saccharomyces cerevisiae”, EMBO J., 6:229-234, 1987.-   Barbas, Kang, Lerner and Benkovic, “Assembly of combinatorial    antibody libraries on phage surfaces: the gene III site,” Proc.    Natl. Acad. Sci. USA, 88(18):7978-7982, 1991.-   Baxter and Jain, “Transport of fluid and macromolecules in tumors,”    Micro. Res., 41:5-23, 1991.-   Beckman, Weiner and Davis, “Antibody Constructs in Cancer Therapy”,    Cancer, 109(2):170-179, 2006.-   Benjamin, Golijanin, Itin, Pode and Keshet, “Selective ablation of    immature blood vessels in established human tumors follows vascular    endothelial growth factor withdrawal,” J. Clin. Invest.,    103(2):159-165, 1999.-   Berman, Mellis, Pollock, Smith, Suh, Heinke, Kowal, Surti, Chess,    Cantor, et al., “Content and organization of the human Ig VH locus:    definition of three new VH families and linkage to the Ig CH locus,”    EMBO J., 7(3):727-738, 1988.-   Borgstrom, Hillan, Sriramarao, Ferrara, “Complete inhibition of    angiogenesis and growth of microtumors by anti-vascular endothelial    growth factor neutralizing antibody: novel concepts of angiostatic    therapy from intravital videomicroscopy,” Cancer Res.,    56(17):4032-1439, 1996.-   Borgstrom Bourdon, Hillan, Sriramarao, Ferrara, “Neutralizing    anti-vascular endothelial growth factor antibody completely inhibits    angiogenesis and growth of human prostate carcinoma micro tumors in    vivo,” Prostate, 35(1):1-10, 1998.-   Borgstrom, Gold, Hillan, Ferrara, “Importance of VEGF for breast    cancer angiogenesis in vivo: implications from intravital microscopy    of combination treatments with an anti-VEGF neutralizing monoclonal    antibody and doxorubicin,” Anticancer Research, 19(5B):4203-11,    1999.-   Bornstein, “Thrombospondins: structure and regulation of    expression,” FASEB J, 6(14):3290-3299, 1992.-   Brekken, Huang, King, Thorpe, “Vascular endothelial growth factor as    a marker of tumor endothelium,” Cancer Res., 58(9):1952-1959, 1998.-   Brekken, Overholser, Stasny, Waltenberger, Minna, Thorpe, “Selective    inhibition of VEGFR2 activity by a monoclonal anti-VEGF antibody    blocks tumor growth in mice”, Cancer Res., 60:5117-24, 2000.-   Brem, “Angiogenesis antagonists: current clinical trials,”    Angiogenesis, 2: 9-20, 1998.-   Brinster, Chen, Trumbauer, Yagle, Palmiter, “Factors Affecting the    Efficiency of Introducing Foreign DNA into Mice by Microinjecting    Eggs”, Proc. Natl. Acad. Sci. USA, 82(13):4438-4442, 1985.-   Burke, Carle, Olson, “Cloning of large segments of exogenous DNA    into yeast by means of artificial chromosome vectors”, Science, 236,    806-812, 1987.-   Burke, Lehmann-Bruinsma, Powell, “Vascular endothelial growth factor    causes endothelial proliferation after vascular injury,” Biochem.    Biophys. Res. Comm., 207:348-354, 1995.-   Burrows and Thorpe, “Vascular targeting-a new approach to the    therapy of solid tumors,” Pharmacol. Ther., 64:155-174, 1994.-   Burrows and Thorpe, “Eradication of large solid tumors in mice with    an immunotoxin directed against tumor vasculature,” Proc. Natl.    Acad. Sci. USA, 90:8996-9000, 1993.-   Burrows, Watanabe, Thorpe, “A murine model for antibody-directed    targeting of vascular endothelial cells in solid tumors,” Cancer    Res., 52:5954-5962, 1992.-   Carmeliet, Ferreira, Breier, Pollefeyt, Kieckens, Gertsenstein,    Fahrig, Vandenhoeck, Harpal, Eberhardt, Declercq, Pawling, Moons,    Collen, Risau, Nagy, “Abnormal blood vessel development and    lethality in embryos lacking a single VEGF allele,” Nature,    380(6573):435-439, 1996.-   Carillo and Lipton, “The Multiple Sequence Alignment Problem in    Biology”, SIAM J. Applied Math., 48:1073, 1988.-   Cheng, Huang, Nagane, Ji, Wang, Shih, Arap, Huang, Cavenee,    “Suppression of glioblastoma angiogenicity and tumorigenicity by    inhibition of endogenous expression of vascular endothelial growth    factor,” Proc. Natl. Acad. Sci. USA, 93:8502-8507, 1996.-   Claffey, Brown, del Aguila, Tognazzi, Yeo, Manseau, Dvorak,    “Expression of vascular permeability factor/vascular endothelial    growth factor by melanoma cells increases tumor growth,    angiogenesis, and experimental metastasis,” Cancer Res., 56:172-181,    1996.-   Clapp, Martial, Guzman, Fentier-Delure, Weiner, “The 16-kilodalton    N-terminal fragment of human prolactin is a potent inhibitor of    angiogenesis,” Endocrinology, 133(3):1292-1299, 1993.-   Clauss, Weich, Breier, Knies, Röckl, Waltenberger, Risau, “The    vascular endothelial cell growth factor receptor Flt-1 mediates    biological activities,” J. Biol. Chem., 271(30):17629-17634, 1996.-   Cohen, Gaskins, Nasoff, “Generation of a Monoclonal Antibody Agonist    to Toll-Like Receptor 4”, Hybridoma, 24(1):27-35, 2005.-   Condeelis and Pollard, “Macrophages: Obligate Partners for Tumor    Cell Migration, Invasion, and Metastasis”, Cell, 124:263-266, 2006.-   Connolly, Heuvelman, Nelson, Olander, Eppley, Delfino, Siegel,    Leimgruber, Feder, “Tumor vascular permeability factor stimulates    endothelial cell growth and angiogenesis,” J. Clin. Invest.,    84:1470-1478, 1989.-   Coughlin, Salhany, Wysocka, Aruga, Kurzawa, Chang, Hunter, Fox,    Trinchieri, Lee, “Interleukin-12 and interleukin-18 synergistically    induce murine tumor regression which involves inhibition of    angiogenesis,” J. Clin. Invest., 101(6):1441-1452, 1998.-   Cullen, Gray, Wilson, Hayenga, Lamsa, Rey, Norton, Berka,    “Controlled Expression and Secretion of Bovine Chymosin in    Aspergillus nidulans”, BioTechnology, 5:369, 1987.-   D'Amato, Loughnan, Flynn, Folkman, “Thalidomide is an inhibitor of    angiogenesis,” Proc. Natl. Acad. Sci. USA, 91(9):4082-4085, 1994.-   D'Angelo, Struman, Martial, Weiner, “Activation of mitogen-activated    protein kinases by vascular endothelial growth factor and basic    fibroblast growth factor in capillary endothelial cells is inhibited    by the antiangiogenic factor 16-kDa N-terminal fragment of    prolactin,” Proc. Natl. Acad. Sci. USA, 92(14):6374-6378, 1995.-   Davies and Cohen, “Interactions of protein antigens with    antibodies,” Proc Natl. Acad. Sci. U.S.A. 93:7-12, 1996. Davies,    Padlan, Sheriff, “Antibody-antigen complexes,” Annu. Rev. Biochem.    59:439-473, 1990.-   Davies and Riechmann, “Antibody VH domains as small recognition    units”, Biotechnology (NY), 13:475-479, 1995.-   Davis and Yancopoulos, “The angiopoietins: Yin and Yang in    angiogenesis”, Curr. Top. Microbiol. Immunol., 237:173-85, 1999.-   Detmar, Brown, Claffey, Yeo, Kocher, Jackman, Berse, Dvorak,    “Overexpression of vascular permeability factor/vascular endothelial    growth factor and its receptors in psoriasis,” J. Exp. Med.,    180:1141-1146, 1994.-   Devereux, Haeberli, Smithies, “A Comprehensive Set of Sequence    Analysis Programs for the VAX”, Nucleic Acids Res., 12:387, 1984.-   DeVore, Hellerqvist, Wakefield, Wamil, Thurman, Minton, Sundell,    Yan, Carter, Wang, York, Zhang, Johnson, “Phase I Study of the    Antineovascularization Drug CM101,” Clin. Cancer Res., 3(3):365-372,    1997.-   deVries, Escobedo, Ueno, Houck, Ferrara, Williams, “The fms-like    tyrosine kinase, a receptor for vascular endothelial growth factor,”    Science, 255(5047):989-991, 1992.-   Dvorak, Nagy, Dvorak, “Structure of solid tumors and their    vasculature: implications for therapy with monoclonal antibodies,”    Cancer Cells, 3:77-85, 1991a.-   Dvorak, Sioussat, Brown, Berse, Nagy, Sotrel, Manseau, Vandewater,    Senger, “Distribution of vascular permeability factor (vascular    endothelial growth factor) in tumors—concentration in tumor blood    vessels,” J. Exp. Med., 174:1275-1278, 1991b.-   Ferrara, “The role of vascular endothelial growth factor in    pathological angiogenesis,” Breast Cancer Res. Treat., 36:127-137,    1995.-   Ferrara, Clapp, Weiner, “The 16K fragment of prolactin specifically    inhibits basal or fibroblast growth factor stimulated growth of    capillary endothelial cells,” Endocrinology, 129(2):896-900, 1991.-   Ferrara, Houck, Jakeman, Winer, Leung, “The vascular endothelial    growth factor family of polypeptides,” J. Cell. Biochem.,    47:211-218, 1991.-   Ferrara, Carver-Moore, Chen, Dowd, Lu, O'Shea, Powell-Braxton,    Hillan, Moore, “Heterozygous embryonic lethality induced by targeted    inactivation of the VEGF gene,” Nature, 380(6573):439-442, 1996.-   Fidler and Ellis, “The implications of angiogenesis for the biology    and therapy of cancer metastasis [comment],” Cell, 79(2):185-188,    1994.-   Fidler, Kumar, Bielenberg, Ellis, “Molecular determinants of    angiogenesis in cancer metastasis,” Cancer J. Sci. Am., 4 Suppl    1:S58-66, 1998.-   Finberg, Knipe, Kurt-Jones, “Herpes Simplex Virus and Toll-Like    Receptors”, Viral Immunol., 18(3):457-465, 2005.-   Folkman and Shing, “Angiogenesis,” J. Biol. Chem., 267:10931-10934,    1992.-   Folkman, Langer, Linhardt, Haudenschild, Taylor, “Angiogenesis    inhibition and tumor regression caused by heparin or a heparin    fragment in the presence of cortisone,” Science, 221:719-725, 1983.-   Fong, Rossant, Gertsenstein, Breitman, “Role of the Flt-1 receptor    tyrosine kinase in regulating the assembly of vascular endothelium,”    Nature, 376:66-70, 1995.-   Forsythe, Jiang, Iyer, Agani, Leung, Koos, Semenza, “Activation of    vascular endothelial growth factor gene transcription by    hypoxia-inducible factor 1,” Mol. Cell. Biol., 16:4604-4613, 1996.-   Fotsis, Zhang, Pepper, Adlercreutz, Montesano, Nawroth, Schweigerer,    “The endogenous oestrogen metabolite 2-methoxyoestradiol inhibits    angiogenesis and suppresses tumour growth,” Nature,    368(6468):237-239, 1994.-   Frank, Hubner, Breier, Longaker, Greenhalgh, Werner, “Regulation of    vascular endothelial growth factor expression in cultured growth    factor expression in cultured keratinocytes. Implications for normal    and impaired wound healing,” J. Biol. Chem., 270:12607-12613, 1995.-   Frankel, “Genetically Engineered Toxins”, Editor Arthur E. Frankel,    Marcel Dekker Inc., New York, N.Y., 1992.-   Frater-Schroder, Risau, Hallmann, Gautschi, Bohlen, “Tumor necrosis    factor type alpha, a potent inhibitor of endothelial cell growth in    vitro, is angiogenic in vivo,” Proc. Natl. Acad. Sci. USA,    84(15):5277-5281, 1987.-   Frazier, “Thrombospondins,” Curr. Opin. Cell Biol., 3(5):792-799,    1991.-   Frische, Meldal, Werdelin, Mouritsen, Jensen, Galli-Stampino, Bock,    “Multiple, Column Synthesis of a Library of T-Cell Stimulating    Tn-Antigenic Glycopeptide Analogues for the Molecular    Characterization of T-Cell-Glycan Specificity”, J. Pept. Sci., 2(4):    212-22, 1996.-   Gabrilovich, D. I., Velders, M. P., Sotomayor, E. M. & Kast, W. M.    (2001). Mechanism of immune dysfunction in cancer mediated by    immature Gr-1+ myeloid cells. J Immunol, 166, 5398-406.-   Gagliardi, Hadd, Collins, “Inhibition of angiogenesis by suramin,”    Cancer Res., 52(18):5073-5075, 1992.-   Gagliardi and Collins, “Inhibition of angiogenesis by    antiestrogens,” Cancer Res., 53(3):533-535, 1993.-   Gagliardi, Kassack, Kreimeyer, Muller, “Antiangiogenic and    antiproliferative activity of suramin analogues,” Cancer Chemother.    Pharmacol., 41(2):117-124, 1998.-   Gagnon, Bielenberg, Gechtman, Miao, Takashima, Soker, Klagsbrun,    “Identification of a natural soluble neuropilin-1 that binds    vascular endothelial growth factor: In vivo expression and antitumor    activity”, Proc. Natl. Acad. Sci. USA, 97(6):2573-2578, 2000.-   Gerber, Condorelli, Park, Ferrara, “Differential transcriptional    regulation of the two vascular endothelial growth factor receptor    genes,” J. Biol. Chem., 272:23659-23667, 1997.-   Gerber, Vu, Ryan, Kowalski, Werb, Ferrara, “VEGF couples    hypertrophic cartilage remodeling, ossification and angiogenesis    during endochondral bone formation”; Nature Medicine, 5(6):623-8,    1999.-   Giovarelli, Cappello, Formi, Salcedo, Moore, LeFleur, Nardelli, Di    Carlo, Lollini, Ruben, Ullrich, Garotta, Musiam, “Tumor rejection    and immune memory elicited by locally released LEC chemokine are    associated with an impressive recruitment of APCs, lymphocytes, and    granulocytes”, J. Immunol., 164, 3200-3206, 2000.-   Glennie, McBride, Worth, Stevenson, “Preparation and performance of    bispecific F(ab′ gamma)2 antibody containing thioether-linked Fab′    gamma fragments,” J. Immunol., 139:2367-2375, 1987.-   Goeddel, “Gene Expression Technology: Methods in Enzymology 185,    Academic Press, San Diego, Calif., 1990.-   Good, Polyerini, Rastinejad, Beau, Lemons, Frazier, Bouck, “A tumor    suppressor-dependent inhibitor of angiogenesis is immunologically    and functionally indistinguishable from a fragment of    thrombospondin,” Proc. Natl. Acad. Sci. USA, 87(17):6624-6628, 1990.-   Goswami, Sahai, Wyckoff, Cammer, Cox, Pizley, Stanley, Segall and    Condeelis, “Macrophages Promote the Invasion of Breast Carcinoma    Cells via a Colony-Stimulating Factor-1/Epidermal Growth Factor    Paracrine Loop”, Cancer Res., 65(12):5278-5283, 2005.-   Grant, Caballero, Bush, Spoerri, “Fibronectin fragments modulate    human retinal capillary cell proliferation and migration,” Diabetes,    47(8):1335-1340, 1998.-   Guo, Jia, Song, Warren, Donner, “Vascular endothelial cell growth    factor promotes tyrosine phosphorylation of mediators of signal    transduction that contain SH2 domains,” J. Biol. Chem.,    270:6729-6733, 1995.-   Hamers-Casterman and Atarhouch, “Naturally Occurring antibodies    Devoid of Light Chains”, Nature, 363(6428):446-448, 1993.-   Hammer, Pursel, Rexroad, Wall, Bolt, Ebert, Palmiter, Brinster,    “Production of Transgenic Rabbits, Sheep and Pigs by    Microinjection”, Nature, 315:680-683, 1985.-   Hanahan and Folkman, “Patterns and emerging mechanisms of the    angiogenic switch during tumorigenesis,” Cell, 86(3):353-364, 1996.-   Harada, Mitsuyama, Yoshida, Sakisaka, Taniguchi, Kawaguchi,    Ariyoshi, Saiki, Sakamoto, Nagata, Sata, Matsuo, Tanikawa, “Vascular    endothelial growth factor in patients with rheumatoid arthritis”,    Scandinavian J. Rheumatol., 27(5):377-80, 1998.-   Haran, Maretzek, Goldberg, Horowitz, Degani, “Tamoxifen enhances    cell death in implanted MCF7 breast cancer by inhibiting endothelium    growth,” Cancer Res., 54(21):5511-5514, 1994.-   Hasselaar and Sage, “SPARC antagonizes the effect of basic    fibroblast growth factor on the migration of bovine aortic    endothelial cells,” J. Cell Biochem., 49(3):272-283, 1992.-   Harlow and Lane, “Antibodies: a Laboratory Manual”, Cold Spring    Harbor Laboratory, Cold Spring Harbor Laboratory, ISBN    978-087969314-5:1-726, 1988.-   Hellerqvist, Thurman, Page, Wang, Russell, Montgomery, Sundell,    “Antitumor effects of GBS toxin: a polysaccharide exotoxin from    group B beta-hemolytic streptococcus,” J. Cancer Res. Clin. Oncol.,    120(1-2):63-70, 1993.-   Henikoff and Henikoff, “Amino acid Substitution Matrices from    Protein Blocks”, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992.-   Hinnen, Hicks, Fink, “Transformation of Yeast”, Proc. Natl. Acad.    Sci. USA, 75:1929, 1978.-   Hiratsuka, Minowa, Kuno, Noda, Shibuya, “Flt-1 lacking the tyrosine    kinase domain is sufficient for normal development and angiogenesis    in mice,” Proc. Natl. Acad. Sci. USA, 95(16):9349-9354, 1998.-   Hiscox and Jiang, “Interleukin-12, an emerging anti-tumour    cytokine,” In Vivo, 11(2):125-132, 1997.-   Holash, Maisonpierre, Compton, Boland, Alexander, Zagzag,    Yancopoulos, Wiegand, “Vessel Cooption, Regression, and Growth in    Tumors Mediated by Angiopoietins and VEGF”, Science, 284:1994-1998,    1999.-   Holliger and Hudson, “Engineered Antibody Fragments and the Rise of    Single Domains”, Nature Biotechnology, 23(9):1126-1136, 2005.-   Holm, “Dali: a Network Tool for Protein Structure Comparison”,    Trends in Biochemical Sciences, 20:478-480, 1995.-   Holm, “Protein Structure Comparison by Alignment of Distance    Matrices”, J. Mol. Biol., 233:123-38, 1993-   Holm, “Touring Protein Fold Space With Dali/FSSP”, Nucleic Acid    Res., 26:316-9, 1998.-   Hood and Granger, “Protein kinase G mediates vascular endothelial    growth factor-induced Raf-1 activation and proliferation in human    endothelial cells,” J. Biol. Chem., 273(36):23504-23508, 1998.-   Hood, Meininger, Ziche, Granger, “VEGF upregulates ecNOS message,    protein, and NO production in human endothelial cells,” Am. J.    Physiol., 274(3 Pt 2):H1054-1058, 1998.-   Hori, Hu, Yasui, Smither, Gresham, Fan, “Differential effects of    angiostatic steroids and dexamethasone on angiogenesis and cytokine    levels in rat sponge implants,” Br. J. Pharmacol., 118(7):1584-1591,    1996.-   Horsmans, Berg, Desager, Mueller, Schott, Fletcher, Steffy, Bauman,    Kerr, Averett, “Isatoribine, an agonist of TLR7, reduces plasma    virus concentration in chronic hepatitis C infection,” Hepatology,    42(3):724-31, 2005. Houben-weyl, Methods of Organic Chemistry,    ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart, 1987.-   Houck, Ferrara, Winer, Cachianes, L1, Leung, “The vascular    endothelial growth factor family: Identification of a fourth    molecular species and characterization of alternative splicing of    RNA,” Mol. Endocrinol., 5(12):1806-1814, 1991.-   Huang, Molema, King, Watkins, Edgington, Thorpe, “Tumor infarction    in mice by antibody-directed targeting of tissue factor to tumor    vasculature,” Science, 275:547-550, 1997.-   Hurwitz, Fehrenbacher, Novotny, Cartwright, Hainsworth, Heim,    Berlin, Baron, Griffing, Holmgren, Ferrara, Fyfe, Rogers, Ross,    Kabbinavar, “Bevacizumab plus Irinotecan, Fluorouracil, and    Leucovorin for Metastatic Colorectal Cancer”, N. Engl. J. Med.,    350:2335-2342, 2004.-   Huse, Sastry, Iverson, Kang, Alting-Mees, Burton, Benkovic and    Lerner, “Generation of a Large Combinatorial Library of the    Immunoglobulin Repertoire in Phage Lambda, Science,    246(4935):1275-1281, 1989.-   Hussain, Kotz, Minasian, Premkumar, Sarosy, Reed, Zhai, Steinberg,    Raggio, Oliver, Figg, Kohn, “Phase II Trial of Carboxyamidotriazole    in Patients With Relapsed Epithelial Ovarian Cancer”, J. Clin.    Oncol., 21(23):4356-4363, 2003.-   Ingber, Fujita, Kishimoto, Sudo, Kanamaru, Brem, Folkman,    “Angioinhibins: Synthetic analogues of fumagillin which inhibit    angiogenesis and suppress tumor growth,” Nature, 48:555-557, 1990.-   Inoue, Itoh, Ueda, Naruko, Kojima, Komatsu, Doi, Ogawa, Tamura,    Takaya, Igaki, Yamashita, Chun, Masatsugu, Becker, Nakao, “Vascular    endothelial growth factor (VEGF) expression in human coronary    atherosclerotic lesions: possible pathophysiological significance of    VEGF in progression of atherosclerosis”, Circulation,    98(20):2108-16, 1998.-   Ito, Fukuda, Murata, Kimura, “Transformation of Intact Yeast Cells    Treated with Alkali Cations”, J. Bacteriol., 153:163-168, 1983.-   Iwamoto, Nomizu, Yamada, Ito, Tanaka, Sugioka, “Inhibition of    angiogenesis, tumour growth and experimental metastasis of human    fibrosarcoma cells HT1080 by a multimeric form of the laminin    sequence Tyr-Ile-Gly-Ser-Arg (YIGSR),” Br. J. Cancer, 73(5):589-595,    1996.-   Jackson, Volpert, Bouck, Linzer, “Stimulation and inhibition of    angiogenesis by placental proliferin and proliferin-related    protein,” Science, 266(5190):1581-1584, 1994.-   Jendraschak and Sage, “Regulation of angiogenesis by SPARC and    angiostatin: implications for tumor cell biology,” Semin. Cancer    Biol., 7(3):139-146, 1996.-   Kabat, Wu, Perry, Gottesman, Foeller, “Sequences of Proteins of    Immunological Interest”, 5th Ed. Public Health Service, National    Institutes of Health, Bethesda, Md., 647-669, 1991.-   Kamphaus, Colorado, Panka, Hopfer, Ramchandran, Torre, Maeshima,    Mier, Sukhatme, and Kalluri, “Canstatin, a Novel Matrix-derived    Inhibitor of Angiogenesis and Tumor Growth”, J. Biol. Chem.,    275(2):1209-1215, 2000.-   Kang, Barbas, Janda, Benkovic and Lerner, “Linkage of Recognition    and Replication Functions by Assembling Combinatorial Antibody Fab    Libraries Along Phage Surfaces”, Proc. Natl. Acad. Sci., U.S.A.,    88(10):4363-4366, 1991.-   Kaufman, Murtha, Davies, “Translational Efficiency of Polycistronic    Mrnas and Their Utilization to Express Heterologous Genes in    Mammalian Cells”, EMBO J., 6:187-195, 1987.-   Kaur, Brat, Devi and Van Meir, “Vasculostatin, a proteolytic    fragment of Brain Angiogenesis Inhibitor 1, is an antiangiogenic and    antitumorigenic factor”, Oncogene, 24:3632-3642, 2005.-   Keck, Hauser, Krivi, Sanzo, Warren, Feder, Connolly, “Vascular    permeability factor, an endothelial cell mitogen related to PDGF,”    Science, 246:1309-1312, 1989.-   Kendall and Thomas, “Inhibition of vascular endothelial cell growth    factor activity by an endogenously encoded soluble receptor,” Proc.    Natl. Acad. Sci. USA, 90:10705-10709, 1993.-   Kenyon, Browne, D'Amato, “Effects of thalidomide and related    metabolites in a mouse corneal model of neovascularization,” Exp.    Eye Res., 64(6):971-978, 1997.-   Kerbel, Viloria-Petit, Okada, Rak, “Establishing a link between    oncogenes and tumor angiogenesis,” Mol. Med., 4(5):286-295, 1998.-   Keyt, Nguyen, Berleau, Duarte, Park, Chen, Ferrara, “Identification    of vascular endothelial growth factor determinants for binding KDR    and FLT-1 receptors. Generation of receptor-selective VEGF variants    by site-directed mutagenesis,” J. Biol. Chem., 271(10):5638-46,    1996.-   Kim, Li, Houck, Winer, Ferrara, “The vascular endothelial growth    factor proteins: identification of biologically relevant regions by    neutralizing monoclonal antibodies,” Growth Factors, 7:53-64, 1992.-   Kim, Li, Winer, Armanini, Gillett, Phillips, “Inhibition of vascular    endothelial growth factor-induced angiogenesis suppresses tumour    growth in vivo,” Nature, 362:841-844, 1993.-   Kim, Kwak, Ahn, So, Liu, Koh, Koh, “Molecular cloning and    characterization of a novel angiopoietin family protein,    angiopoietin-3”, FEBS Lett., 443(3):353-6, 1999.-   Kipriyanov, Kupriyanova, Little, Moldenhauer, “Rapid detection of    recombinant antibody fragments directed against cell-surface    antigens by flow cytometry”, J. Immunol. Meth., 196:51-62, 1996.-   Kipriyanov, Moldenhauer, Little, “High level production of soluble    single chain antibodies in small-scale Escherichia coli    cultures”, J. Immunol. Meth., 200:69-77, 1997.-   Kiss, Fisher, Pesavento, Dai, Valero, Ovecka, Nolan, Phipps,    Velappan, Chasteen, Martinez, Waldo, Pavlik, Bradbury, “Antibody    binding loop insertions as diversity elements”, Nucleic Acids    Research, 34(19):e132, 2006.-   Kleinman, Weeks, Schnaper, Kibbey, Yamamura, Grant, “The laminins: a    family of basement membrane glycoproteins important in cell    differentiation and tumor metastases,” Vitam. Horm., 47:161-186,    1993.-   Kondo, Asano, Suzuki, “Significance of vascular endothelial growth    factor/vascular permeability factor for solid tumor growth, and its    inhibition by the antibody,” Biochem. Biophys. Res. Commun,    194(3):1234-1241, 1993.-   Korpelainen and Alitalo, “Signaling angiogenesis and    lymphangiogenesis,” Curr. Opin. Cell Biol., 10(2):159-164, 1998.-   Kremer, Breier, Risau, Plate, “Up-regulation of flk-1/vascular    endothelial growth factor receptor 2 by its ligand in a cerebral    slice culture system,” Cancer Res., 57:3852-3859, 1997.-   Kroll and Waltenberger, “The vascular endothelial growth factor    receptor KDR activates multiple signal transduction pathways in    porcine aortic endothelial cells”, J. Biol. Chem., 272:32521-7,    1997.-   Kroll and Waltenberger, “VEGF-A induces expression of eNOS and iNOS    in endothelial cells via VEGF receptor-2 (KDR),” Biochem. Biophys.    Res. Commun., 252(3):743-746, 1998.-   Kurjan and Herskowitz, “Structure of a Yeast Pheromone Gene (MFα): a    Putative α-Factor Precursor Contains Four Tandem Copies of mature    α-Factor”, Cell, 30:933-943, 1982.-   Kyte and Doolittle, “A simple method for displaying the hydropathic    character of a protein,” J. Mol. Biol., 157(1):105-132, 1982.-   Lane, Iruela-Arispe, Sage, “Regulation of gene expression by SPARC    during angiogenesis in vitro. Changes in fibronectin,    thrombospondin-1, and plasminogen activator inhibitor-1,” J. Biol.    Chem., 267(23):16736-16745, 1992.-   Le Gall, Reusch, Little and Kipriyanov, “Effect of Linker Sequences    Between the Antibody Variable Domains on the Formation, Stability    and Biological Activity of a Bispecific Tandem Diabody”, Protein    Engineering, Design & Selection, 17(4):357-366, 2004.-   Lee, Clapp, Martial, Weiner, “Inhibition of urokinase activity by    the antiangiogenic factor 16K prolactin: activation of plasminogen    activator inhibitor 1 expression,” Endocrinology, 139(9):3696-3703,    1998.-   Lewis and Pollard, “Distinct Role of Macrophages in Different Tumor    Microenvironments”, Cancer Res., 66(2):605-612, 2006.-   Lin, Sankar, Shan, Dewhirst, Polyerini, Quinn, Peters, “Inhibition    of tumor growth by targeting tumor endothelium using a soluble    vascular endothelial growth factor receptor,” Cell Growth Differ.,    9:49-58, 1998.-   Lin, Buxton, Acheson, Radziejewski, Maisonpierre, Yancopoulos,    Channon, Hale, Dewhirst, George, Peters, “Anti-angiogenic gene    therapy targeting the endothelium-specific receptor tyrosine kinase    Tie2”, Proc. Natl. Acad. Sci., USA, 95(15):8829-34, 1998.-   Lin, Nguyen, Mendoza, Escandon, Fei, Meng, Modi, “Preclinical    pharmacokinetics, interspecies scaling, and tissue distribution of a    humanized monoclonal antibody against vascular endothelial growth    factor”, J. Pharmacol. Exp. Therap., 288(1):371-8, 1999.-   Lin, Nguyen, Russell and Pollard, “Colony-Stimulating Factor 1    Promotes Progression of Mammary Tumors to Malignancy”, J. Exp. Med.,    193(6):727-739, 2001.-   Lin, Li, Gnatovskiy, Deng, Zhu, Grzesik, Qian, Xue and Pollard,    “Macrophages Regulate the Angiogenic Switch in a Mouse Model of    Breast Cancer”, Cancer Res., 66(23):11238-11246, 2006.-   Lindner and Borden, “Effects of tamoxifen and interferon-beta or the    combination on tumor-induced angiogenesis,” Int. J. Cancer,    71(3):456-461, 1997.-   Lingen, Polyerini, Bouck, “Retinoic acid and interferon alpha act    synergistically as antiangiogenic and antitumor agents against human    head and neck squamous cell carcinoma,” Cancer Res.,    58(23):5551-5558, 1998.-   Lingen, Polyerini, Bouck, “Inhibition of squamous cell carcinoma    angiogenesis by direct interaction of retinoic acid with endothelial    cells,” Lab. Invest., 74(2):476-483, 1996.-   Lin-ke, Hong-Qu, Nagy, Eckelhoefer, Masse, Dvorak, Dvorak, “Vascular    targeting of solid and ascites tumours with antibodies to vascular    endothelial growth factor,” Eur. J. Cancer, 32A(14):2467-2473, 1996.-   Loupakis, Falcone, Masi, Fioravanti, Kerbel, Del Tacca, Bocci    “Vascular Endothelial Growth Factor Levels in Immunodepleted plasma    of Cancer Patients As a Possible Pharmacodynamic Marker for    Bevacizumab Activity,” J. Clin. One, 1816-1818, 2007.-   Luckow and Summers, “High Level Expression of Nonfused Foreign Genes    with Autographa californica Nuclear Polyhedrosis Virus Expression    Vectors”, Virology, 170:31-39, 1989.-   Luo, Toyoda, Shibuya, “Differential inhibition of fluid accumulation    and tumor growth in two mouse ascites tumors by an antivascular    endothelial growth factor/permeability factor neutralizing    antibody,” Cancer Res., 58(12):2594-2600, 1998a.-   Luo, Yamaguchi, Shinkai, Shitara, Shibuya, “Significant expression    of vascular endothelial growth factor/vascular permeability factor    in mouse ascites tumors,” Cancer Res., 58(12):2652-2660, 1998b.-   Majewski, Skopinska, Marczak, Szmurlo, Bollag, Jablonska “Vitamin D3    is a potent inhibitor of tumor cell-induced angiogenesis,” J.    Investig. Dermatol. Symp. Proc., 1(1):97-101, 1996.-   Malecaze, Clamens, Simorre-Pinatel, Mathis, Chollet, Favard, Bayard,    Plouet, “Detection of vascular endothelial growth factor messenger    RNA and vascular endothelial growth factor-like activity in    proliferative diabetic retinopathy,” Arch. Ophthalmol.,    112:1476-1482, 1994.-   Maisonpierre, Suri, Jones, Bartunkova, Wiegand, Radziejewski,    Compton, McClain, Aldrich, Papadopoulos, Daly, Davis, Sato,    Yancopoulos, “Angiopoietin-2, a natural antagonist for Tie2 that    disrupts in vivo angiogenesis,” Science, 277(5322):55-60, 1997.    Manetti, Cappello, Botta, Corelli, Mongelli, Biasoli, Borgia,    Ciomei, “Synthesis and binding mode of heterocyclic analogues of    suramin inhibiting the human basic fibroblast growth factor,”    Bioorg. Med. Chem., 6(7):947-958, 1998.-   Massey, “Catalytic Antibodies Catching On”, Nature, 328:457-458,    1987.-   Mazure, Chen, Yeh, Laderoute, Giaccia, “Oncogenic transformation and    hypoxia synergistically act to modulate vascular endothelial growth    factor expression,” Cancer Res., 56:3436-3440, 1996.-   McNamara, Harmey, Walsh, Redmond, Bouchier-Hayes, “Significance of    angiogenesis in cancer therapy [published erratum appears in Br J.    Surg., October; 85(10):1449, 1998,” Br. J. Surg., 85(8):1044-1055.    1998.-   Merrifield, “Solid Phase Peptide Synthesis 1. Synthesis of a    Tetrapeptide”, J. Am. Chem. Assoc., 85:2149-2154, 1964.-   Mesiano, Ferrara, Jaffe, “Role of vascular endothelial growth factor    in ovarian cancer: inhibition of ascites formation by    immunoneutralization,” Am. J. Pathol., 153(4):1249-1256, 1998.-   Millauer, Longhi, Plate, Shawver, Risau, Ullrich, Strawn,    “Dominant-negative inhibition of Flk-1 suppresses the growth of many    tumor types in vivo,” Cancer Res., 56:1615-1620, 1996.-   Mills, Brooker and Camerini-Otero, “Sequences of human    immunoglobulin switch regions: implications for recombination and    transcription,” Nucl. Acids Res., 18:7305-7316, 1990.-   Moore, Arenberg, Addison, Keane, Streiter, “Tumor angiogenesis is    regulated by CXC chemokines,” J. Lab. Clin. Med., 132(2):97-103,    1998.-   Mordenti, Thomsen, Licko, Chen, Meng, Ferrara, “Efficacy and    concentration-response of murine anti-VEGF monoclonal antibody in    tumor-bearing mice and extrapolation to humans”, Toxicologic    Pathology, 27(1):14-21, 1999.-   Morrison, Wims, Kobrin and Oi, “Production of novel immunoglobulin    molecules by gene transfection,” Mt. Sinai J. Med., 53(3):175, 1986.-   Muller, Li, Christinger, Wells, Cunningham, De Vos, “Vascular    Endothelial growth factor: Crystal structure and functional mapping    of the kinase domain receptor binding site”, Proc. Natl. Acad. Sci.    USA., 94:7192-7197, 1997.-   Muller, Chen, Christinger, Li, Cunningham, Lowman, de Vos, “VEGF and    the Fab fragment of a humanized neutralizing antibody: crystal    structure of the complex at 2.4 A resolution and mutational analysis    of the interface,” Structure, 6(9):1153-67, 1998.-   Mustonen and Alitalo, “Endothelial receptor tyrosine kinases    involved in angiogenesis,” J. Cell Biol., 129:895-898, 1995.-   Myers and Miller, “Optical Alignments in Linear Space”, CABIOS,    4:11-17, 1988.-   Nagashima, Yoshino, Aono, Takai, Sasano, “Inhibitory effects of    anti-rheumatic drugs on vascular endothelial growth factor in    cultured rheumatoid synovial cells”, Clin. Exp. Immunol.,    116(2):360-5, 1999.-   Nagler, Feferman, Shoshan, “Reduction in basic fibroblast growth    factor mediated angiogenesis in vivo by linomide,” Connect Tissue    Res., 37(1-2):61-68, 1998.-   Nakamura Voller and Bidwell, “Enzyme Immunoassays: Heterogeneous and    Homogeneous Systems”, In: Handbook of Experimental Immunology, Vol.    1: Immunochemistry, D. M. Weir (Ed.), Blackwell Scientific    Publications, Oxford 1986, Chapter 27.-   Needleman and Wunsch, “A General Method Applicable to the Search For    Similarities in the Amino Acid Sequence of Two Proteins”, J. Mol.    Biol., 48:443, 1970.-   Neuberger and Milstein, “Somatic hypermutation,” Curr. Opin.    Immunol., 7:248-254, 1995.-   Neufeld, Cohen, Gengrinovitch, Poltorak, “Vascular endothelial    growth factor (VEGF) and its receptors,” FASEB J., 13(1):9-22, 1999.-   Nicaise, Valerio-Lepiniec, Minard, Desmadril, “Affinity transfer by    CDR grafting on a nonimmunoglobulin scaffold”, Protein Sci., 13:    1882-1891, 2004.-   Niida, Kaku, Amano, Yoshida, Kataoka, Nishikawa, Tanne, Maeda,    Nishikawa, Kodama, “Vascular endothelial growth factor can    substitute for macrophage colony-stimulating factor in the support    of osteoclastic bone resorption”, J. Exp. Med., 190(2):293-8, 1999.-   Nozaki, Sakurai, Raisler, Baffi, Witta, Ogura, Brekken, Sage,    Ambati, Ambati, “Loss of SPARC-mediated VEGFR-1 suppression after    injury reveals a novel antiangiogenic activity of VEGF-A”, J. Clin.    Invest., 116(2):422-9, 2006.-   Oikawa, Hirotani, Nakamura, Shudo, Hiragun, Iwaguchi, “A highly    potent antiangiogenic activity of retinoids,” Cancer Lett.,    48(2):157-162, 1989.-   Olander, Connolly, DeLarco, “Specific binding of vascular    permeability factor to endothelial cells,” Biochem. Biophys. Res.    Comm., 175:68-76, 1991.-   O'Reilly, Holmgren, Shing, Chen, Rosenthal, Moses, Lane, Cao, Sage,    Folkman “Angiostatin: a novel angiogenesis inhibitor that mediates    the suppression of metastases by a Lewis lung carcinoma,” Cell,    79:315-328, 1994.-   O'Reilly, Boehm, Shing, Fukai, Vasios, Lane, Flynn, Birkhead, Olsen,    Folkman “Endostatin: an endogenous inhibitor of angiogenesis and    tumor growth,” Cell, 88(2):277-285, 1997.-   Palmiter and Brinster, “Transgenic Mice”, Cell, 41:343-345, 1985.-   Palmiter, Norstedt, Gelinas, Hammer, Brinster,    “Metallothionein-Human GH Fusion Genes Stimulate Growth of Mice”,    Science, 222:809-814, 1983.-   Parmley and Smith, “Antibody-selectable filamentous fd phage    vectors: affinity purification of target genes,” Gene,    73(2):305-318, 1988.-   Pearson and Lipman, “Improved tools for biological sequence    analysis”, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988.-   Pearson, “Rapid and sensitive sequence comparison with FASTP and    FASTA”, Methods in Enzymology, 183:63-98, 1990.-   Pepper, Ferrara, Orci, Montesano, “Leukemia inhibitory factor (LIF)    inhibits angiogenesis in vitro,” J. Cell Sci., 108(1):73-83, 1995.-   Petersen, Wang, Yalcin-Chin, Li, Peyton, Minna, Harran, Wang,    “Autocrine TNFα Signaling Renders Human Cancer Cells Susceptible to    Smac-Mimetic-Induced Apoptosis”, Cancer Cell, 12(5):445-456, 2007.-   Petrova, Nykanen, Norrmen, Ivanov, Andersson, Haglund, Puolakkainen,    Wempe, von Melchner, Gradwohl, Vanharanta, Aaltonen, Saharinen,    Gentile, Clarke, Taipale, Oliver, Alitalo, “Transcription factor    PROX1 induces colon cancer progression by promoting the transition    from benign to highly dysplastic phenotype”, Cancer Cell,    13(5):407-19, 2008.-   Pike, Yao, Jones, Chemey, Appella, Sakaguchi, Nakhasi,    Teruya-Feldstein, Wirth, Gupta and Tosato, “Vasostatin, a    Calreticulin Fragment, Inhibits Angiogenesis and Suppresses Tumor    Growth”, J. Exp. Med., 188(12):2349-2356, 1998.-   Pike, Yao, Setsuda, Jones, Chemey, Appella, Sakaguchi, Nakhasi,    Atreya, Teruya-Feldstein, Wirth, Gupta and Tosato, “Calreticulin and    Calreticulin Fragments Are Endothelial Cell Inhibitors That Suppress    Tumor Growth”, Blood, 94(7):2461-2468, 1999.-   Plate, Breier, Weich, Mennel, Risau, “Vascular endothelial growth    factor and glioma angiogenesis: coordinate induction of VEGF    receptors, distribution of VEGF protein and possible in vivo    regulatory mechanisms,” Int. J. Cancer, 59:520-529, 1994.-   Potgens, Westphal, DeWaal, Ruiter, “The role of vascular    permeability factor and basic fibroblast growth factor in tumor    angiogenesis,” In: Growth Factors in Tumor Angiogenesis, Berlin:    Walter de Gruyer & Co. pp. 57-70, 1995.-   Presta, Chen, O'Connor, Chisholm, Meng, Krummen, Winkler, Ferrara,    “Humanization of an anti-vascular endothelial growth factor    monoclonal antibody for the therapy of solid tumors and other    disorders,” Cancer Res., 57:4593-4599, 1997.-   Qiu, Wang, Cai, Wang, Yue, “Small antibody mimetics comprising two    complementarity-determining regions and a framework region for tumor    targetingl, Nature Biotechnology, 25(8): 921-929, 2007-   Quinn, Thurman, Sundell, Zhang, Hellerqvist, “CM101, a    polysaccharide antitumor agent, does not inhibit wound healing in    murine models,” J. Cancer Res. Clin. Oncol., 121(4):253-256, 1995.-   Raychaudhury and D'Amore, “Endothelial cell regulation by    transforming growth factor-beta,” J. Cell Biochem., 47(3):224-229,    1991.-   Reff and Heard, “A Review of Modifications to Recombinant    Antibodies: Attempt to Increase Efficacy in Oncology Applications”,    Critical Reviews in Oncology Hematology, 40:25-35, 2001.-   Reiter, Ulrich Brinkmann, Lee and Pastan, “Engineering Antibody Fv    Fragements for Cancer Detection and Therapy: Disulfide-Stabilized Fv    Fragments”, Nature Biotechnology, 14:1239-1245, 1996.-   Richer and Lo, “Introduction of human DNA into mouse eggs by    injection of dissected human chromosome fragments”, Science,    245:175-177, 1989.-   Ryan, Eppler, Hagler, Bruner, Thomford, Hall, Shopp, O'Neill,    “Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic    humanized monoclonal antibody”, Toxicologic Pathology, 27(1):78-86,    1999.-   Sakamoto, Tanaka, Togho, Ogawa, “Heparin plus cortisone acetate    inhibit tumor growth by blocking endothelial cell proliferation,”    Canc. J., 1:55-58, 1986.-   Saleh, Stacker, Wilks, “Inhibition of growth of C6 glioma cells in    vivo by expression of antisense vascular endothelial growth factor    sequence,” Cancer Res., 56:393-401, 1996.-   Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory    Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,    1989.-   Sang, “Complex role of matrix metalloproteinases in angiogenesis,”    Cell Res., 8(3):171-177, 1998.-   Schultz, Tanner, Hofmann, Emini, Condra, Jones, Kieff, Ellis,    “Expression and Secretion in Yeast of a 400-Kda Envelope    Glycoprotein Derived from Epstein-Barr Virus”, Gene, 54:113-123,    1987.-   Seed, “an LFA-3 Cdna Encodes a Phospholipid-Linked Membrane Protein    Homologous to its Receptor CD2”, Nature, 329:840, 1987.-   Senger, Galli, Dvorak, Perruzzi, Harvey, Dvorak, “Tumor cells    secrete a vascular permeability factor that promotes accumulation of    ascites fluid,” Science, 219:983-985, 1983.-   Senger, Perruzzi, Feder, Dvorak, “A highly conserved vascular    permeability factor secreted by a variety of human and rodent tumor    cell lines,” Cancer Res., 46:5629-5632, 1986.-   Senger, Connolly, Vandewater, Feder, Dvorak, “Purification and    NH2-terminal amino acid sequence of guinea pig tumor secreted    vascular permeability factor,” Cancer Res., 50:1774-1778, 1990.-   Serafini, P., De Santo, C., Marigo, I., Cingarlini, S., Dolcetti,    L., Gallina, G., Zanovello, P. & Bronte, V. (2004). “Derangement of    immune responses by myeloid suppressor cells”, Cancer Immunol    Immunother, 53, 64-72.-   Shalaby, Rossant, Yamaguchi, Gertsenstein, Wu, Breitman, Schuh,    “Failure of blood-island formation and vasculogenesis in    Flk-1-deficient mice,” Nature, 376:62-66, 1995.-   Sheibani and Frazier, “Thrombospondin 1 expression in transformed    endothelial cells restores a normal phenotype and suppresses their    tumorigenesis,” Proc. Natl. Acad. Sci. USA, 92(15):6788-6792, 1995.-   Sheu, Yen, Kan, “Inhibition of angiogenesis in vitro and in vivo:    comparison of the relative activities of triflavin, an    Arg-Gly-Asp-containing peptide and anti-alpha(v)beta3 integrin    monoclonal antibody,” Biochim. Biophys. Acta, 1336(3):445-454, 1997.-   Shibuya, “Vascular endothelial growth factor receptor-1    (VEGFR-1/Flt-1): a dual regulator for angiogenesis”, Angiogenesis,    9(4):225-30, 2007.-   Shojaei, Wu, Malik, Zhong, Baldwin, Schanz, Fuh, Gerber, Ferrara,    “Tumor refractoriness to anti-VEGF treatment is mediated by    CD11b⁺Gr1⁺ myeloid cells”, Nature Biotechnology, 25:911-920, 2007.-   Sideras, Mizuta, Kanamori, Suzuki, Okamoto, Kuze, Ohno, Doi,    Fukuhara, Hassan, et al., “Production of sterile transcripts of C    gamma genes in an IgM-producing human neoplastic B cell line that    switches to IgG-producing cells,” Intl. Immunol., 1(6):631-642,    1989.-   Siemeister, Martiny-Baron, Marme, “The pivotal role of VEGF in tumor    angiogenesis: molecular facts and therapeutic opportunities,” Cancer    Metastasis Rev., 17(2):241-248., 1998.-   Sinkar, White, Gordon, “Molecular Biology of Ri-Plasmid a    Review”, J. Biosci (Bangalore), 11:47-58, 1987.-   Sioussat, Dvorak, Brock, Senger, “Inhibition of vascular    permeability factor (vascular endothelial growth factor) with    antipeptide antibodies,” Arch. Biochem. Biophys., 301:15-20, 1993.-   Sipos, Tamargo, Weingart, Brem, “Inhibition of tumor angiogenesis,”    Ann. NY Acad. Sci., 732:263-272, 1994.-   Smith and Waterman, “Comparison of Biosequences”, Adv. Appl. Math.,    2:482, 1981.-   Smith, Summers, Fraser, “Production of Human Beta Interferon in    Insect Cells Infected With Baculovirus Expression Vector”, Mol.    Cell. Biol., 3:2156-2165, 1983.-   Soff, Sanderowitz, Gately, Verrusio, Weiss, Brem, Kwaan, “Expression    of plasminogen activator inhibitor type 1 by human prostate    carcinoma cells inhibits primary tumor growth, tumor-associated    angiogenesis, and metastasis to lung and liver in an athymic mouse    model,” J. Clin. Invest., 96(6):2593-2600, 1995.-   Soker, Takashima, Miao, Neufeld, Klagsbrun., “Neuropilin-1 is    expressed by endothelial and tumor cells as an isoform-specific    receptor for vascular endothelial growth factor,” Cell,    92(6):735-745, 1998.-   Springer, Chen, Kraft, Bednarski, Blau, “VEGF gene delivery to    muscle: potential role for vasculogenesis in adults,” Mol. Cell,    2(5):549-558, 1998.-   Stella and Himmelstein, “Prodrugs: A chemical approach to targeted    drug delivery”, Directed Drug Delivery, Borchardt et al., Eds. Human    Press, 1985, pp 247-267.-   Sweetwyne, Brekken, Workman, Bradshaw, Carbon, Siadak, Murri and    Sage, “Functional Analysis of the Matricellular Protein SPARC with    Novel Monoclonal Antibodies”, J. Histochem. Cytochem.,    52(6):723-733, 2004.-   Tada, Fukunaga, Wakabayashi, Masumi, Sato, Izumi, Kohno, Kuwano,    “Inhibition of tubular morphogenesis in human microvascular    endothelial cells by co-culture with chondrocytes and involvement of    transforming growth factor beta: a model for avascularity in human    cartilage,” Biochim. Biophys. Acta, 1201(2):135-142, 1994.-   Takano, Gately, Neville, Herblin, Gross, Engelhard, Perricone,    Eidsvoog, Brem, “Suramin, an anticancer and angiosuppressive agent,    inhibits endothelial cell binding of basic fibroblast growth factor,    migration, proliferation, and induction of urokinase-type    plasminogen activator,” Cancer Res., 54(10):2654-2660, 1994.-   Tanaka, Manome, Wen, Kufe, Fine, “Viral vector-mediated transduction    of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor    growth,” Nat. Med., 3(4):437-442, 1997.-   Terman, Dougher-Vermazen, Carrion, Dimitrov, Armellino,    Gospodarowicz, Bohlen, “Identification of the KDR tyrosine kinase as    a receptor for vascular endothelial cell growth factor,” Biochem.    Biophys. Res. Comm., 187:1579-1586, 1992.-   Terman, Khandke, Dougher-Vermazan, Maglione, Lassam, Gospodarowicz,    Persico, Bohlen, Eisinger, “VEGF receptor subtypes KDR and FLT1 show    different sensitivities to heparin and placenta growth factor,”    Growth Factors, 11(3):187-195, 1994.-   Thomas, “Vascular endothelial growth factor, a potent and selective    angiogenic agent,” J. Biol. Chem., 271:603-606, 1996.-   Thompson, Higgins, Gibson, “CLUSTAL W: Improving the sensitivity of    progressive multiple sequence alignment through sequence weighting,    position-specific gap penalties and weight matrix choice”, Nucleic    Acids Res., 22:4673-4680, 1994.-   Thorpe, Derbyshire, Andrade, Press, Knowles, King, Watson, Yang,    Rao-Bette, “Heparin-Steroid Conjugates New Angiogenesis Inhibitors    with Antitumor Activity in Mice,” Cancer Res., 53:3000-3007, 1993.-   Tischer, Mitchell, Hartman, Silva, Gospodarowicz, Fiddes, Abraham,    “The human gene for vascular endothelial growth factor,” J. Biol.    Chem., 266:11947-11954, 1991.-   Tolsma, Volpert, Good, Frazier, Polyerini, Bouck, “Peptides derived    from two separate domains of the matrix protein thrombospondin-1    have anti-angiogenic activity,” J. Cell Biol., 122(2):497-511, 1993.-   Tryggvason, “The laminin family,” Curr. Opin. Cell Biol.,    5(5):877-882, 1993.-   Valenzuela, Griffiths, Rojas, Aldrich, Jones, Zhou, McClain,    Copeland, Gilbert, Jenkins, Huang, Papadopoulos, Maisonpierre,    Davis, Yancopoulos, “Angiopoietins 3 and 4: diverging gene    counterparts in mice and humans”, Proc. Natl. Acad. Sci., USA,    96(5):1904-9, 1999.-   van den Beucken, Neer, Sablon, Desmet, Celis, Hoogenboom, Hufton,    “Building novel binding ligands to B7.1 and B7.2 based on human    antibody single variable light chain domains”, J. Mol. Biol.,    310:591-601, 2001.-   van dijk, Warnaar, van Eendenburg, Thienpont, Braakman, Boot,    Fleuren and Bolhuis, “Induction of tumor-cell lysis by bi-specific    monoclonal antibodies recognizing renal-cell carcinoma and CD3    antigen,” Int. J. Cancer, 43:344-349, 1989.-   Varfolomeev, Blankenship, Wayson, Fedorova, Kayagaki, Garg, Zobel,    Dynek, Elliott, Wallweber, Flygare, Fairbrother, Deshayes, Dixit,    Vucic, “IAP Antagonists Induce Autoubiquitination of c-IAPs, NF-κB    Activation, and TNFα-Dependent Apoptosis”, Cell, 131 (4): 669-681,    2007.-   Vince, Wong, Khan, Feltham, Chau, Ahmed, Benetatos, Chunduru,    Condon, McKinlay, Brink, Leverkus, Tergaonkar, Schneider, Callus,    Koentgen, Vaux, Silke, “IAP Antagonists Target cIAP1 to Induce    TNFα-Dependent Apoptosis”, Cell, 131(4):682-693, 2007.-   Volpert, Lawler, Bouck, “A human fibrosarcoma inhibits systemic    angiogenesis and the growth of experimental metastases via    thrombospondin-1,” Proc. Natl. Acad. Sci. USA, 95(11):6343-6348,    1998.-   Vukanovic, Passaniti, Hirata, Traystman, Hartley-Asp, Isaacs,    “Antiangiogenic effects of the quinoline-3-carboxamide linomide,”    Cancer Res., 53(8):1833-1837, 1993.-   Wagner, Milstein, Neuberger, “Codon bias targets mutation,” Nature,    376:732, 1995.-   Waltenberger, Claesson-Welsh, Siegbahn, Shibuya, Heldin, “Different    signal transduction properties of KDR and Flt1, two receptors for    vascular endothelial growth factor,” J. Biol. Chem.,    269(43):26988-26995, 1994.-   Waltenberger, Mayr, Pentz, Hombach, “Functional upregulation of the    vascular endothelial growth factor receptor KDR by hypoxia,”    Circulation, 94:1647-1654, 1996.-   Waltenberger, Mayr, Frank, Hombach, “Suramin is a potent inhibitor    of vascular endothelial growth factor. A contribution to the    molecular basis of its antiangiogenic action,” J. Mol. Cell    Cardiol., 28(7):1523-1529, 1996.-   Wamil, Thurman, Sundell, DeVore, Wakefield, Johnson, Wang,    Hellerqvist, “Soluble E-selectin in cancer patients as a marker of    the therapeutic efficacy of CM101, a tumor-inhibiting    anti-neovascularization agent, evaluated in phase I clinical    trail,” J. Cancer Res. Clin. Oncol., 123(3):173-179, 1997.-   Ward, Güssow, Griffiths, Jones, Winter, “Binding Activities of a    Repertoire of Single Immunoglobulin Variable Domains Secreted from    Escherichia coli”, Nature, 341(6242):544-546, 1989.-   Wells, “Starving cancer into submission”, Chem. Biol., 5(4):R87-88,    1998.-   Whitehurst, Flister, Bagaitkar, Volk, Bivens, Pickett,    Castro-Rivera, Brekken, Gerard, Ran, “Anti-VEGF-A therapy reduces    lymphatic vessel density and expression of VEGFR-3 in an orthotopic    breast tumor model”, Int. J. Cancer, 121(10):2181-91, 2007.-   Wiesmann, Fuh, Christinger, Eigenbrot, Wells, de Vos, “Crystal    structure at 1.7 A resolution of VEGF in complex with domain 2 of    the Flt-1 receptor,” Cell, 91(5):695-704, 1997.-   Willman et al., “Prodrugs in cancer therapy”, Biochem. Soc. Trans.,    14:375-382, 1988.-   Winter and Milstein, “Man-made antibodies,” Nature, 349:293-299,    1991.-   Wolff, Guerin, Laterra, Bressler, Indurti, Brem, Goldstein,    “Dexamethasone inhibits glioma-induced formation of capillary like    structures in vitro and angiogenesis in vivo,” Klin. Padiatr.,    209(4):275-277, 1997.-   Wyckoff, Wang, Lin, Wang, Pixley, Stanley, Graf, Pollard, Segall and    Condeelis, “A Paracrine Loop Between Tumor Cells and Macrophages is    Required for Tumor Cell Migration in Mammary Tumors”, Cancer Res.,    64:7022-7029, 2004.-   Xie, Chen, Fu, Harter, Young, Sunkara, Novak, Villanueva-Siles,    Ratech, “Podoplanin (d2-40): a new immunohistochemical marker for    reactive follicular dendritic cells and follicular dendritic cell    sarcomas”, Int. J. Clin. Exp. Pathol., 1(3):276-84, 2008.-   Yoon, Yoo, Choi, Do, Kang, Lee, Azuma, Kim, “Inhibitory effect of    Korean mistletoe (Viscum album coloratum) extract on tumour    angiogenesis and metastasis of haematogenous and non-haematogenous    tumour cells in mice,” Cancer Lett, 97(1):83-91, 1995.-   Yoshida, Kaneko, Tsukamoto, Han, Ichinose, Kimura, “Suppression of    hepatoma growth and angiogenesis by a fumagillin derivative TNP470:    possible involvement of nitric oxide synthase,” Cancer Res.,    58(16):3751-3756, 1998.-   Young, MacKenzie, Narang, Oomen and Baenziger, “Thermal    Stabilization of a Single-Chain Fv Antibody Fragment by Introduction    of a Disulphide Bond”, FEBS Letters, 16396(377):135-139, 1995.-   Yamamura, Kibbey, Jun, Kleinman, “Effect of Matrigel and laminin    peptide YIGSR on tumor growth and metastasis,” Semin. Cancer Biol.,    4(4):259-265, 1993.-   Zachary, “Vascular endothelial growth factor: how it transmits its    signal,” Exp. Nephrol., 6(6):480-487, 1998.-   Zambryski, Herrera-Estreila, DeBlock, Van Montagu, Schell “Genetic    Engineering, Principles and Methods”, Hollaender and Setlow (eds.),    Vol. VI, pp. 253-278, Plenum Press, New York, 1984.-   Zapata, Ridgway, Mordenti, Osaka, Wong, Bennett, Carter,    “Engineering Linear F(Ab′)₂ Fragments For Efficient Production in    Escherichia Coli and Enhanced Antiproliferative Activity”, Protein    Eng., 8(10):1057-1062, 1995.-   Zhang, Gildersleeve, Yang, Xu, Loo, Uryu, Wong, Schultz, “A New    Strategy for the Synthesis of Glycoproteins”, Science, 303(5656):    371-373, 2004.-   Ziche, Donnini, Morbidelli, Parenti, Gasparini, Ledda, “Linomide    blocks angiogenesis by breast carcinoma vascular endothelial growth    factor transfectants,” Br. J. Cancer, 77(7):1123-1129, 1998.-   Zou, Anisowicz, Hendrix, Thor, Neveu, Sheng, Rafidi, Seftor, Sager,    “Maspin, a serpin with tumor-suppressing activity in human mammary    epithelial cells”, Science, 28:263(5146):526-9, 1994.

What is claimed is:
 1. A method of inhibiting angiogenesis, comprisingadministering an antibody that binds to VEGF, or an immunoconjugatethereof, to an animal in an amount effective to inhibit angiogenesis insaid animal wherein the antibody that binds to VEGF comprises at leastone heavy chain variable region that comprises three CDRs and at leastone light chain variable region that comprises three CDRs, wherein saidantibody comprises: (a) a variable light (VL) CDR1 having the amino acidsequence of SEQ ID NO:8, a VL CDR2 having the amino acid sequence of SEQID NO:9, a VL CDR3 having the amino acid sequence of SEQ ID NO:10, avariable heavy (VH) CDR1 having the amino acid sequence of SEQ ID NO:5,a VH CDR2 having the amino acid sequence of SEQ ID NO:6, and a VH CDR3having the amino acid sequence of SEQ ID NO:7; or (b) a light chainvariable region that has the amino acid sequence of SEQ ID NO:4, or aheavy chain variable region that has the amino acid sequence of SEQ IDNO:3.
 2. The method of claim 1, wherein said animal has cancer.
 3. Themethod of claim 2, wherein said cancer is selected from the groupconsisting of lung, breast, ovary, colorectal, kidney, bladder, smallcell carcinomas and glioblastomas.
 4. The method of claim 1, whereinsaid animal has a disease associated with corneal, retinal or choroidalneovascularization.
 5. The method of claim 1, wherein said animal has anocular neovascular disease, macular degeneration, age-related maculardegeneration, diabetic retinopathy, neovascular glaucoma, arthritis,rheumatoid arthritis, atherosclerosis, thyroid hyperplasia, Grave'sdisease, hemangioma or psoriasis.
 6. The method of claim 1, wherein saidanimal is a human subject.
 7. The method of claim 1, wherein saidantibody is attached to at least a first therapeutic agent.
 8. Themethod of claim 7, wherein the at least a first therapeutic agent isselected from the group consisting of radiotherapeutic agent,chemotherapeutic agent, anti-angiogenic agent, apoptosis-inducing agent,anti-tubulin drug, anti-cellular or cytotoxic agent, cytokine, chemokineand coagulant.
 9. The method of claim 1, further comprisingadministering a second therapeutic agent to said animal.
 10. The methodof claim 9, wherein said second therapeutic agent is a chemotherapeuticagent.
 11. The method of claim 9, wherein said second therapeutic agentis selected from the group consisting of pyrimidine analogs, platinumcoordination complexes, camptothecins, tyrosine kinase inhibitors andinhibitors of receptor tyrosine kinases.
 12. The method of claim 9,wherein said second therapeutic agent is 5-fluorouracil, capecitabine,oxaliplatin, cisplatin, carboplatin, irinotecan, axitinib, sunitinib, orpaclitaxel.
 13. The method of claim 1, wherein said antibody comprisessaid VL CDR1 having the amino acid sequence of SEQ ID NO:8, said VL CDR2having the amino acid sequence of SEQ ID NO:9, said VL CDR3 having theamino acid sequence of SEQ ID NO:10, said VH CDR1 having the amino acidsequence of SEQ ID NO:5, said VH CDR2 having the amino acid sequence ofSEQ ID NO:6, and said VH CDR3 having the amino acid sequence of SEQ IDNO:7.
 14. The method of claim 1, wherein said antibody comprises saidlight chain variable region that has the amino acid sequence of SEQ IDNO:4.
 15. The method of claim 1, wherein said antibody comprises saidheavy chain variable region that has the amino acid sequence of SEQ IDNO:3.
 16. The method of claim 1, wherein said antibody comprises saidlight chain variable region that has the amino acid sequence of SEQ IDNO:4 and said heavy chain variable region that has the amino acidsequence of SEQ ID NO:3.
 17. The method of claim 1, wherein saidantibody comprises the amino acid sequence of SEQ ID NO:21.
 18. Themethod of claim 1, wherein said antibody is a fully human antibody. 19.The method of claim 1, wherein said antibody is a whole antibodycomprising an antibody constant region.
 20. The method of claim 19,wherein said antibody comprises a heavy chain that has the amino acidsequence of SEQ ID NO:24 and a light chain that has the amino acidsequence of SEQ ID NO:25.
 21. The method of claim 1, wherein saidantibody is an antigen binding fragment of an antibody.
 22. The methodof claim 1, wherein said antibody has a binding affinity for VEGF thatcorresponds to a Kd of less than 10 nM when said antibody is in IgGformat.
 23. A method for inhibiting angiogenesis in the treatment of ananimal that has a disease characterized by excessive or pathologicalangiogenesis, said method comprising administering an antibody thatbinds to VEGF, or an immunoconjugate thereof, to said animal in anamount effective to inhibit said angiogenesis, thereby treating saiddisease in said animal wherein the antibody that binds to VEGF comprisesat least one heavy chain variable region that comprises three CDRs andat least one light chain variable region that comprises three CDRs,wherein said antibody comprises: (a) a variable light (VL) CDR1 havingthe amino acid sequence of SEQ ID NO:8, a VL CDR2 having the amino acidsequence of SEQ ID NO:9, a VL CDR3 having the amino acid sequence of SEQID NO:10, a variable heavy (VH) CDR1 having the amino acid sequence ofSEQ ID NO:5, a VH CDR2 having the amino acid sequence of SEQ ID NO:6,and a VH CDR3 having the amino acid sequence of SEQ ID NO:7; or (b) alight chain variable region that has the amino acid sequence of SEQ IDNO:4, or a heavy chain variable region that has the amino acid sequenceof SEQ ID NO:3.
 24. The method of claim 23, wherein said disease iscancer.
 25. The method of claim 24, wherein said cancer is selected fromthe group consisting of lung, breast, ovary, colorectal, kidney,bladder, small cell carcinomas and glioblastomas.
 26. The method ofclaim 23, wherein said disease is associated with corneal, retinal orchoroidal neovascularization.
 27. The method of claim 23, wherein saiddisease is ocular neovascular disease, macular degeneration, age-relatedmacular degeneration, diabetic retinopathy, neovascular glaucoma,arthritis, rheumatoid arthritis, atherosclerosis, thyroid hyperplasia,Grave's disease, hemangioma or psoriasis.
 28. The method of claim 23,wherein said animal is a human subject.
 29. The method of claim 23,wherein said antibody is attached to at least a first therapeutic agent.30. The method of claim 29, wherein the at least a first therapeuticagent is selected from the group consisting of radiotherapeutic agent,chemotherapeutic agent, anti-angiogenic agent, apoptosis-inducing agent,anti-tubulin drug, anti-cellular or cytotoxic agent, cytokine, chemokineand coagulant.
 31. The method of claim 23, further comprisingadministering a second therapeutic agent to said animal.
 32. The methodof claim 24, further comprising administering a second therapeutic agentto said animal, wherein said second therapeutic agent is an anti-canceragent.
 33. The method of claim 32, wherein said second therapeutic agentis an anti-cancer agent selected from the group consisting of pyrimidineanalogs, platinum coordination complexes, camptothecins, tyrosine kinaseinhibitors and inhibitors of receptor tyrosine kinases.
 34. The methodof claim 32, wherein said second anti-cancer agent is 5-fluorouracil,capecitabine, oxaliplatin, cisplatin, carboplatin, irinotecan, axitinib,sunitinib, or paclitaxel.
 35. The method of claim 23, wherein saidantibody comprises said VL CDR1 having the amino acid sequence of SEQ IDNO:8, said VL CDR2 having the amino acid sequence of SEQ ID NO:9, saidVL CDR3 having the amino acid sequence of SEQ ID NO:10, said VH CDR1having the amino acid sequence of SEQ ID NO:5, said VH CDR2 having theamino acid sequence of SEQ ID NO:6, and said VH CDR3 having the aminoacid sequence of SEQ ID NO:7.
 36. The method of claim 23, wherein saidantibody comprises said light chain variable region that has the aminoacid sequence of SEQ ID NO:4.
 37. The method of claim 23, wherein saidantibody comprises said heavy chain variable region that has the aminoacid sequence of SEQ ID NO:3.
 38. The method of claim 23, wherein saidantibody comprises said light chain variable region that has the aminoacid sequence of SEQ ID NO:4 and said heavy chain variable region thathas the amino acid sequence of SEQ ID NO:3.
 39. The method of claim 23,wherein said antibody comprises the amino acid sequence of SEQ ID NO:21.40. The method of claim 23, wherein said antibody is a fully humanantibody.
 41. The method of claim 23, wherein said antibody is a wholeantibody comprising an antibody constant region.
 42. The method of claim41, wherein said antibody comprises a heavy chain that has the aminoacid sequence of SEQ ID NO:24 and a light chain that has the amino acidsequence of SEQ ID NO:25.
 43. The method of claim 23, wherein saidantibody is an antigen binding fragment of an antibody.
 44. The methodof claim 23, wherein said antibody has a binding affinity for VEGF thatcorresponds to a Kd of less than 10 nM when said antibody is in IgGformat.
 45. A method for treating cancer characterized by excessiveangiogenesis in an animal, said method comprising administering anantibody that binds to VEGF, or an immunoconjugate thereof, to saidanimal in an amount effective to inhibit said angiogenesis, therebytreating said cancer in said animal; wherein the antibody that binds toVEGF comprises at least one heavy chain variable region that comprisesthree CDRs and at least one light chain variable region that comprisesthree CDRs, wherein said antibody comprises: (a) a variable light (VL)CDR1 having the amino acid sequence of SEQ ID NO:8, a VL CDR2 having theamino acid sequence of SEQ ID NO:9, a VL CDR3 having the amino acidsequence of SEQ ID NO:10, a variable heavy (VH) CDR1 having the aminoacid sequence of SEQ ID NO:5, a VH CDR2 having the amino acid sequenceof SEQ ID NO:6, and a VH CDR3 having the amino acid sequence of SEQ IDNO:7; or (b) a light chain variable region that has the amino acidsequence of SEQ ID NO:4, or a heavy chain variable region that has theamino acid sequence of SEQ ID NO:3.